• The resistance to therapy in PDAC is a multifaceted phenomenon mainly driven by intrinsic (within tumor) and extrinsic (acquired) factors.
• The intrinsic factors are inherent mutations, pre-existing epigenetic modifications, tumor microenvironment (TME), hypoxia, epithelial to mesenchymal transition (EMT), cell cycle dysregulation.
• Extrinsic factors are secondary genetic mutations, adaptive changes in TME, phenotypic plasticity, and increased drug detoxification.
• Comprehensive understanding of these resistance mechanisms can aid in strategic development of effective therapeutic approaches including modalities targeting cell cycle regulators, hypoxia, TME, reactivating T cells and restoring anti-tumor immune responses, adoptive cell therapy that uses CAR-T cells to better recognize and attack tumor cells, synergistic combination of immunotherapy and chemotherapy.

Pancreatic ductal adenocarcinoma (PDAC), a common form of pancreatic cancer (PC) accounts for ∼90% of all pancreatic tumors [1,2]. PC is currently the fourth leading cause of cancer related deaths in the US accounting for about 8.3% of all cancer death [3]. Not only does PC incur a significant direct cost from highly specialized treatment due to late-stage diagnosis, but also higher societal economic cost [4-6]. PC has a five-year survival rate of approximately 10%, having one of the worst prognoses among all cancer types. About 80% of patients present with unresectable (metastatic or locally advanced) disease with a median survival of 5 months [7]. Even though targeted therapies have dramatically changed the prognosis of some types of cancer, such as lung and ovarian cancer, PC continues to be a disease with a poor prognosis and few treatment options [8,9].

Several risk factors have been associated with PC which can either be modifiable such as smoking, obesity, diabetes mellitus, dietary factors, and lifestyle choices or non-modifiable namely, age, gender, genetic predisposition and familial syndromes, chronic pancreatitis and inflammatory conditions, microbiota, occupational and environmental exposures. Amongst several risk factors the direct impact of lifestyle such as smoking, heavy alcohol consumption, obesity, and physical inactivity is well-established for the development of PDAC. While the direct relationship between lifestyle factors and treatment resistance in PDAC remains underexplored, it is plausible that the same factors contributing to the development of PDAC could also influence its progression and response to treatment. Further research is needed to elucidate these potential connections and to determine whether lifestyle modifications could enhance treatment efficacy or mitigate resistance [10-12].

The insolvability of this complex disease can be attributed to late diagnosis, scarcity of sensitive and specific biomarkers, early dissemination of metastases, and notably, resistance to chemotherapy, radiotherapy, and currently available targeted therapies. Diagnosis of PC is a significant challenge because of multiple factors namely non-specific symptoms linked with the disease, and closeness to major blood vessels leading to invasion and delay in diagnosis. Therefore, majority of tumors are not resectable when presented [13]. Additionally, screening of unselected populations for PC is not conducted due to low lifetime risk of about 1%. Whilst for population at high-risk, cancer antigen 19-9 (CA 19-9) is the only serum marker approved by the US FDA for use in the routine management of PC [14]. However, this marker is not used for asymptomatic population screening due to its poor positive predictive value. The quest for potential biomarkers including liquid biopsy to facilitate prognosis and treatment of PC has been an area of intense research. In addition, the high incidence of genetic mutation associated in PDAC has led to the investigation of cell free DNA (cf-DNA) and tumor cell DNA (ct-DNA) as a screening or diagnostic test [15,16].

Surgical resection improves 5-year survival to 25%. However, most patients present with metastasized or locally advanced disease preventing resection. Worldwide, rates of resection for Stage I/II disease vary from 35% to 69% [17]. Completion of adjuvant therapy after resection is associated with improved survival, painfully, many patients are unable to complete. Modest chemotherapy benefits are observed in both resectable and non-resectable forms because of drug resistance and 3-4 grade adverse events [17]. Although several chemotherapy regimens have been approved for metastatic PDAC, Gemcitabine remains in the forefront [18]. It is usually used in combination with Nab-Paclitaxel, which improves survival time compared to Gemcitabine monotherapy. The combination regimen of 5-Fluorouracil, Leucovorin, Irinotecan, and Oxaliplatin i.e., FOLFIRINOX has also shown efficacy in metastatic PDAC. Furthermore, FOLFIRINOX offers better disease-free survival (DFS) compared to Gemcitabine (21.6 vs 12.8 months) but is associated with serious adverse effects (75.9 vs 52.9%). For patients with BRCA1/2 or PALB2-mutated PDAC (5-9%) the combination of Gemcitabine and Cisplatin is effective. Olaparib, a PARP inhibitor has shown significant efficacy in BRCA mutated patients [19]. Potentially, patients deficient in DNA mismatch repair (dMMR) with high microsatellite instability (MSI-H) may respond to the immune checkpoint inhibitor, Pembrolizumab. Recently, PDAC patients with NTRK1–3 or ROS1 have demonstrated potential clinical response towards precision medicine approaches by selective tropomyosin receptor kinase (TRK) and ROS1 inhibitors such as larotrectinib and entrectinib [20-21].

PDAC has a weak response to current therapy regimens. There are several reasons why PC is still difficult to treat with conventional therapies e. g. high frequency of genomic alterations leading to significant genomic instability. Given the fact that genetic mutations in PDAC play a major role in tumor initiation and development, researchers have attempted to exploit them as therapeutic targets. However, after several trials, it has been evident that genetic mutations could be used as targets only in a small percentage of patients [22-25]. Numerous clinical trials targeting specific molecular pathways are ongoing, but many have failed. Major reasons for this are either the related drugs are not available or because the mutated genes cannot be targeted by drugs. Several observations implied the participation of non-genetic mechanisms in drug resistance [23,25]. Such resistance is supported by both intrinsic and acquired factors. Therefore, understanding these inherent mechanisms is an essential step to improve efficacy leading to better prognosis.

The resistance to therapy in PDAC is a multifaceted phenomenon mainly driven by two mechanisms such as intrinsic (within tumour) and extrinsic (acquired). The intrinsic factors are inherent mutations, pre-existing epigenetic modifications, tumor microenvironment (TME), hypoxia, epithelial to mesenchymal transition (EMT) and other conditions causing dysregulation of cell cycle that are part of the initial development of the cancer. Extrinsic factors are secondary genetic mutations, adaptive changes in TME, phenotypic plasticity, and increased drug detoxification. Figure 1 illustrates different intrinsic and extrinsic mechanisms responsible for cancer cell rescue against PC therapeutics.

Intrinsic factors

Cell cycle dysregulation in PC: The dysregulation of cell cycle due to aberrant CDK and cyclin activity is frequently observed in various tumors including PC, resulting in unrestrained cell proliferation - one of the critical hallmarks of cancer [26]. Large-scale molecular analyses of PC have confirmed that driver genes such as KRAS, CDKN2A, TP53, and SMAD4 are altered in more than 50% of cases. Intrinsic aberrations like genomic deletions promote hypermethylation, and silencing of CDKN2A converge to dysregulate CDKs, which are key regulators in the progression of PC [27]. The inactivation of p16INK4A, an endogenous inhibitor of the CDK4/6-Cyclin D1 complex, activates this complex, leading to the hyperphosphorylation of the tumor suppressor retinoblastoma (RB) protein. Once hyperphosphorylated, the inactivated E2F transcription factor allows cells to progress through the G1/S transition. Therefore, the aberrant loss of p16INK4A affects the inhibition of CDK4/6, resulting in uncontrolled cell growth. Additionally, mutant KRAS regulates CDK4/6 activity through the canonical RAF/MEK/ERK/MAPK signaling pathway and stimulates the transcriptional activation of CCND1, which encodes Cyclin D1 [28]. Notably, targeting CDK4 has been shown to be synthetically lethal with KRAS mutations in non-small cell lung cancer (NSCLC); however, similar outcomes have yet to be explored in PDAC [29].

Genetic mutations: KRAS, a GTPase with notably prevalent mutations occurring in over 90% of PC in turn contributing to intrinsic resistance by activating downstream signaling pathways namely RAF/MEK/ERK and PI3K/AKT/mTOR [30]. KRAS G12D and G12V are the most common, about 39.2 and 32.5% of all KRAS mutations respectively while KRAS G12C represents only ~1% of PDAC [31]. The oncogenic KRAS is mainly involved in regulation of G1/S transition, TGF-β signaling, integrin signaling, regulation of cell invasion, homophilic cell adhesion and small guanine triphosphate (GTPase)-dependent signaling [32]. These aberrant pathways further promote survival and proliferation, making the cancer particularly resilient to many treatments [33]. Also, mutant KRAS drives metabolic reprogramming, including increased glucose uptake and utilization through aerobic glycolysis (Warburg effect), which supports rapid cancer cell growth and resistance to metabolic stress. The inability to target KRAS has always remained a challenge to develop a drug against non-targetable mutations. Notably, Sotorasib was an approved drug that targets KRAS G12C, however, efforts proved ineffective due to feedback mechanism and activation of the MAPK pathway via complexes like MRAS-SHOC2-PP1C [34]. However, MRTX1133, a selective inhibitor targeting KRAS G12D, has shown significant efficacy in preclinical models and remains hope for the overall therapeutic response in 39% of patients with KRAS G12D-mutated PDAC  [34]. 

TP53 also known as a guardian of the genome is mutated in ~ 50-75% of PDAC cases. Mutated TP53 impairs cell’s ability to undergo apoptosis in response to DNA damage, leading to loss of cell cycle control, evasion of apoptosis, and thus causing intrinsic resistance [23].

Furthermore, mutant CDKN2A is present in about 95% of PDAC cases and encodes for p16INK4a, and p14ARF proteins. p16INK4a is an inhibitor of cyclin-dependent kinase (CDK4/6) while p14ARF activates p53 tumor suppressor. Further, inactivation of CDKN2A leads to loss of cell cycle control, allowing for unrestrained cell proliferation primarily due to aberrant TGF-β signaling contributing to the aggressive nature of PDAC [35]. SMAD4 mutations, appearing in nearly 50% of PDAC cases, perturb TGF-β signaling pathway, which normally inhibits cell growth and promotes apoptosis. The loss of SMAD4 further enhances tumor growth and metastasis, making it a key player in the resistance of PDAC to multiple therapies [36]. BRCA1/2 mutation, although less common, can also contribute to intrinsic resistance by impairing DNA repair mechanisms, leading to genomic instability. DNA damage response ATM pathway is also found to be mutated, albeit at lower frequencies in PDAC.

Overall, the combined effect of these mutations in leading genes result in loss of tumor suppressor functions, induced genetic instability, decreased apoptosis, and reduced response to the therapy. About 63 defined genetic changes in 12 different signaling pathways that are known to be dysregulated in the vast majority of PDAC cases. The complex network of signaling, genetic mutations, crosstalk between PC cells and TME makes PDAC difficult to treat disease [37].

Epigenetic regulators: Epigenetic regulators play critical roles in dysregulating cell cycle and promoting intrinsic resistance. Aberrant epigenetic regulations include histone methylation of cancer-related genes which are involved in abnormal proliferation, cell cycle dysregulation, immune escape, and metabolic programming of tumor cells. p16, a tumor suppressor encoded by CDKN2A, was found to gain de novo methylation in ~20% of different primary neoplasms [38]. Mutations in TP53 and BRCA1, are frequently identified in multiple cancers and the level of methylation is positively associated with tumor size. It is known that hypoacetylation of histone (H3K27ac) and methylation (H3K9me3) are distinctly associated with transcriptional repression of genes involved in apoptosis, DNA damage response leading to aberrant changes in cell cycle [39]. Overexpression of DNMTs and HDACs promotes key changes in DNA methylation and histone acetylation patterns, leading to transcriptional silencing of tumor suppressors and DNA repair genes. These alterations enhance cell survival and resistance to therapy. The multiple epigenetic inhibitors are found to be toxic in clinic, however, it remains to be seen in PC to what extent the selective inhibitors for DNMT1 (GSK3685032), HDAC9 (Nanatinostat) or the HDAC6 (Ricolinostat) exhibit reduced toxicity and improved efficacy [40-43].

Post-transcriptional gene regulation (PTGR): PTGR plays a key role in modulating gene expression and various other processes that contribute to intrinsic resistance. Aberrant RNA splicing events can lead to the production of splice variants that promote resistance to therapy. Moreover, alternative splicing of crucial genes involved in apoptosis, cell cycle regulation, and DNA repair can result in the production of protein isoforms with altered functions that enhance cancer cell survival and resistance. SF3B1 is often mutated in cancers, including PC, leading to aberrant splicing of pre-mRNAs [44]. Such mutations can produce splice variants of genes like BCL2, resulting in isoforms that inhibit apoptosis and confer resistance to chemotherapy. Also, nuclear shuttling is crucial for the regulation of gene expression and the function of RNA-binding proteins (RBPs). Dysregulation of nuclear-cytoplasmic transport can contribute to drug resistance by altering the localization and availability of mRNA transcripts and RBPs. Exportin-1 (XPO1) is a nuclear export receptor that mediates the transport of various cargoes, including mRNAs and proteins. Overexpression of XPO1 in PC cells can lead to the mislocalization of tumor suppressor proteins and therapeutic targets, thus promoting cell proliferation. In PC, alterations in RNA stability mechanisms can lead to the overexpression of oncogenes and the downregulation of tumor suppressor genes, contributing to intrinsic resistance. The RBP HuR (ELAVL1) is known to stabilize mRNAs of oncogenes and growth factors in PC and contribute to cell cycle dysregulation [45]. Overexpression of HuR enhances the stability of mRNAs encoding for proteins like VEGF and COX-2, promoting tumor growth, angiogenesis, and resistance to chemotherapy.

DNA repair mechanisms: DNA repair mechanisms play a crucial role in maintaining genomic integrity and cell survival in PC contributing significantly to intrinsic resistance against various therapies. Homologous recombination (HR) is a high-fidelity DNA repair pathway that repairs double stranded breaks (DSBs) and ensures genomic stability. PC cells often exhibit dysregulated HR leading to enhanced repair of DNA damage induced by chemo and radiotherapy. Increased HR repair capacity is associated with resistance to platinum-based drugs like cisplatin and PARP inhibitors [46]. Non-Homologous end joining (NHEJ) is an error-prone DNA repair pathway that directly rejoins broken DNA ends, often leading to small insertions or deletions and contributes to resistance against DNA-damaging agents. This pathway is crucial for repairing DSBs induced by chemotherapy and ionizing radiation, promoting cell survival and therapeutic resistance [47]. Dysregulation of mismatch repair (MMR) pathway components in PC cells leads to genomic instability and resistance to DNA-damaging therapies. Reduced MMR activity allows cancer cells to tolerate DNA replication errors induced by chemotherapy agents, promoting cell survival and resistance [48]. Base excision repair (BER) corrects small, non-helix-distorting base lesions caused by oxidation, alkylation, or deamination. Enhanced BER pathway activity in PC cells contributes to resistance against oxidative stress and chemotherapy-induced DNA damage [49]. Dysregulation of Fanconi anemia (FA) pathway components in PC cells leads to impaired repair of interstrand crosslinks (ICLs) induced by platinum-based chemotherapy. The reduced FA pathway activity confers resistance to crosslinking agents by preventing efficient removal of DNA lesions and promoting cell survival [50].

Key regulators of cell cycle: CDKs are serine/threonine kinases that regulate cell cycle progression and various critical cellular functions. Regulatory mechanisms for CDKs include post-transcriptional modifications, phosphorylation, protein folding, and subcellular localization [51]. CDKs are constitutively expressed, while cyclin levels fluctuate with the cell cycle stages. Cyclin D regulation involves the Ras/RAF/MEK/ERK signaling pathway and GSK3β-mediated degradation. CDK4/6-cyclin D, CDK2-cyclin E and CDK2-cyclin A complexes target the RB protein during G1 to S phase transition by inhibiting E2F. The CDK-cyclin interaction network is complex, with functional redundancy where CDK2 can substitute for CDK4/6, and CDK1 can substitute for other CDKs. CDK1 is the only essential CDK in the cell cycle; its absence prevents embryonic development past the morula and blastocyst stages, highlighting its crucial role in driving cell division [52].

The dysregulation of CDK and cyclin activity in cancer leads to unchecked cell proliferation, a hallmark of malignancy. PDAC exemplifies this, with frequent mutations in KRAS, TP53, and CDKN2A promoting CDK-mediated cell cycle progression. Dysregulation of the CDK4/6-cyclin D-Rb pathway in PDAC enhances proliferation, while mutations in tumor suppressors like p16INK4a compromise cell cycle checkpoints, fostering oncogenic transformation. Additionally, CDK5 collaborates with mutant KRAS to promote PDAC progression. Targeting CDKs, including CDK4/6 and CDK9, shows promise in pre-clinical settings, potentially inducing senescence or apoptosis in cancer cells [53]. The mechanisms underlying intrinsic resistance to CDK4/6 are being tested in pre-clinical and clinical studies using combination strategies with other signaling pathway inhibitors. Moreover, the interplay between CDKs and DNA damage pathways underscores their critical role in maintaining genomic stability and suggests avenues for therapeutic intervention in PC [54].

In PC, there is a pressing need for treatment alternatives, where the inhibition of CDKs represents a possible approach against this difficult-to-treat disease. There are few CDK inhibitors, which represented targeted therapeutic strategies in PDAC and were aimed at disrupting cell cycle regulation thereby potentially enhancing the efficacy of existing treatments or providing new avenues for therapy [55]. SNS-032 is selective CDK9 inhibitor that has been evaluated in preclinical studies for PDAC in combination with Gemcitabine. Preclinical data suggests potential synergy between SNS-032 and mTOR inhibitors in promoting apoptosis possibly through metabolic reprogramming and inhibition of survival pathways [54]. Riviciclib, CDK1, CDK4, and CDK9 inhibitor, could sensitize PC cells to Gemcitabine-induced apoptosis simultaneously targeting CDKs and Akt/mTOR signaling leading to inhibition of both tumor progression and angiogenesis. Further, it also decreases the gene and protein expression of the antiapoptotic protein COX-2, concomitant with reduced p8 gene expression, while simultaneously increasing gene and protein expression of the proapoptotic protein BNIP3. The efficacy of Riviciclib highlights its potential as a therapeutic agent in combating PC by targeting critical pathways involved in tumor progression and resistance mechanisms [56]. Palbociclib (PD-0332991), Ribociclib (LEE-011), and Abemaciclib (LY2835219) are the CDK4/6 inhibitors that are known to prevent RB phosphorylation, arresting cells in G1 phase, inhibiting DNA synthesis and cancer cell growth. These selective inhibitors were investigated for their ability to induce apoptosis and sensitize pancreatic tumors to chemotherapy, particularly in RB-positive cases. Currently, there are no specific combination trials of these inhibitors explicitly focused on PDAC. However, these CDK4/6 inhibitors have been extensively studied in various cancers, including breast cancer, where they are approved and used in combination with other therapies [54]. Dinaciclib (SCH727965) is a pan CDK inhibitor namely targeting CDK1, CDK2, CDK5, and CDK9. This inhibitor is mechanistically known to block cell cycle progression, reduce RB phosphorylation, inhibits relax activity downstream of oncogenic KRAS. Dinaciclib has demonstrated efficacy in reducing growth, migration, and colony formation of PC cells in preclinical models. Dinaciclib has been evaluated in Gemcitabine combination for PDAC where it was shown to enhance the cytotoxic effects of Gemcitabine in preclinical studies potentially overcoming resistance mechanisms in PDAC cells [57]. 

Figure 2 represents the landscape of different inhibitors used for targeting CDKs. However, patients tend to become insensitive to treatment or acquire drug resistance. The possible mechanisms of acquired resistance to CDK4/6 inhibitors in PC are (1) alteration of cyclin D-CDK4/6-Rb pathway (2) activation of other pathways, including the FGFR1 or PI3K/AKT/mTOR pathway (3) modulation of the TME by CDK4/6 inhibitors due to changes in PD-L1 expression. Comprehensive understanding of possible mechanisms of intrinsic and acquired resistance would be necessary to provide potential combinatory strategies with other inhibitors of compensatory signaling pathways to overcome drug resistance [58].

Hypoxia induced acidosis: Hypoxia, a common feature of pancreatic TME, significantly contributes to intrinsic resistance to therapy. In PDAC, there is a decrease in tissue partial oxygen pressure, with median pO2 0–5.3 mmHg (0-0.7%) compared to nearby normal tissue at pO2 24.3–92.7 mmHg (3.2–12.3%). The inadequate vasculature in pancreatic tumors leads to regions of low oxygen tension that cause HIF-1α and HIF-2α to stabilize and translocate to the nucleus, where they dimerize with HIF-1β and activate the transcription of target genes involved in angiogenesis, metabolism, and survival. Importantly, hypoxia also upregulates carbonic anhydrase (mainly CA IX and XII), which helps cells adapt to acidic conditions generated by hypoxia and contributes to chemoresistance [59]. Also, HIFs activate anti-apoptotic proteins like Bcl-2, stimulates EMT through HIF-1α-mediated upregulation of Snail and Twist, which repress epithelial marker E-cadherin and promote mesenchymal markers like N-cadherin and Vimentin mainly mediated through activation of NF-κB. Additional activated genes include those necessary for sustaining energy production in the cells, which mainly occurs via the activation of glycolysis, inhibition of oxidative phosphorylation, and by increasing the expression of glucose transporters namely GLUTs to enable a higher uptake of this nutrient. The other metabolic enzymes upregulated due to hypoxia are carbonic anhydrases (CAs) and indoleamine 2,3 dioxygenase (IDO). These enzymes contribute to an acidic and tryptophan depleted TME respectively, which suppresses immune cell function. The resulting TME is nutrient-poor and highly acidic, creating conditions unfavorable for cytotoxic T cells while promoting the survival and activity of immunosuppressive cells including regulatory T cells (Treg) and M2 polarized macrophages which facilitate tumor progression. Another important factor contributing to acidic TME is the Warburg effect. The increased glucose metabolism leads to a reduction in the pH of the microenvironment because of lactate secretion. An acid-mediated invasion hypothesis suggests that H+ ions secreted by cancer cells diffuse into the surrounding stroma, modifying the tumor-stroma interface to promote increased invasiveness. The Warburg effect is believed to be an early event in tumorigenesis arising as a direct consequence of initial oncogenic mutations such as KRAS in PC. Further, hypoxia has also been demonstrated to enhance autophagy through HIF-1?, sustaining the survival of PDAC tumor cells, especially those that are poorly differentiated. Moreover, the interaction of HIF-1α with NOTCH signaling inhibit PDAC terminal cell differentiation. This interaction is believed to contribute to cancer cell ‘stemness’ within the hypoxic niches of TME [60]. This process not only enhances tumor cell invasiveness but also causes resistance by promoting a more aggressive tumor phenotype [59]. Furthermore, hypoxia leads to intrinsic drug resistance through the upregulation of drug efflux pumps partly due to HIF-1α-mediated transcription of ABC transporters and other drug resistance mechanisms [60].

A second major canonical inducible regulator of hypoxia response is HIF-2α. With respect to PC, in depth study revealed a significant interaction between HIF-2α and Wnt/β-catenin which enhances β-catenin activity and stabilizes HIF-2α. It has been found to drive PC proliferation, promote metabolic reprogramming, induce stemness and facilitate EMT. Recent studies have also implemented HIF-2α expression in cancer associated fibroblast (CAFs) with tumor growth and progression. This expression is also liked with recruitment of immunosuppressive immune cells into TME [59-61].

Additionally, myeloid derived suppressor cells (MDSCs) further contribute to the immune suppression by depleting essential amino acids required for T-cell metabolism, impairing T-cell tracking through L-selectin cleavage, and inhibiting T-cell activation via increased PD-L1 expression. Hypoxic conditions also lead to an accumulation of Treg cells which prevent effective T-cell activation through cytokine release and elevated CTLA-4 expression. Therefore, for effective immunotherapy in PDAC and other solid tumors must address immunosuppressive effects of hypoxia-induced alterations in TME [59].

Hypoxia induced carbonic anhydrases in PDAC: Carbonic anhydrases (CA) are ubiquitous metalloenzymes that catalyze CO2–HCO3- interconversions thereby providing buffering system across cell compartments and in the extracellular space [61]. When tumor cells transition to an aerobic glycolysis, there is a significant pH difference between the extracellular and intracellular pH (pHe and pHi, respectively). Export of metabolic acids in the extracellular space ultimately lowers pHe. In most normal cells, a differential is maintained between pHi and pHe such that the extracellular space is at a slightly more basic environment (pHe: 7.3) relative to the intracellular environment (pHi: 7.2). In hypoxic tumor cells, however, pHe drops to values between 6.5 to 7.1 with negligible decrease in pHi, majorly facilitated by CAs [62]. This provides benefit for tumor cell survival and growth, dissemination, and invasion. There are 16 CA isoforms expressed in humans, 12 are catalytically active. These isoforms have distinct characteristic in terms of cellular distribution, physiology, and function. Out of 15 isoforms, CA I, II, III, VII, VIII, X, XI, and XIII are expressed in the cytoplasm, CA VA and VB are in the mitochondria and CA VI is secretory, and rest isoforms CA IV (GPI anchored), IX, XII, and XIV are membrane-bound [63]. The membrane-bound isoforms are highly active enzymes, out of these only CAIX and CAXII have been associated with tumorigenesis, invasion, and metastasis [64]. The expression of CAIX is under the control of HIF-1, is primarily located in hypoxic tumor core [65]. CAIX is expressed in a wide range of tumor types, including PC, colorectal, lung, breast, cervical cancer, glioblastoma, and is linked to poor prognosis and drives migration and invasion by both catalytic and non-catalytic mechanisms [63,66,67]. Moreover, CAIX expression is limited to a few healthy tissues, such as intrahepatic biliary ducts, duodenum, and gastric mucosa, highlighting its potential for developing cancer-targeted therapies [66]. Due to this very reason, CAIX has been a target of choice for several years including small molecules, targeted therapy, or more recently chimeric antigen receptors (CARs) based cell therapy. A CAIX selective small molecule SLC-0111 has recently progressed to phase Ib/II clinical study [64]. Interestingly, we have also shown striking potency against PDAC of Methazolamide (MZM), a synthetic derivative of sulphonamide currently approved for the treatment of increased intraocular pressure (IOP) in chronic open-angle glaucoma, secondary glaucoma and preoperatively in acute angle-closure glaucoma against PC. We could clearly demonstrate synergistic activity of MZM in combination with Gemcitabine at both in vitro and in vivo levels along with significant reduction in expression of stem cell markers namely RAC-1, OCT4, CD34, CD14, Nanog and proliferation marker (Ki-67). In addition, anti-angiogenic potential, namely wound-healing, tube formation and expression of important anti-angiogenic markers like HIF-1α, VEGF were profoundly modulated in MZM and combination group. Figure 3 depicts pharmacological inhibition of CAIX using MZM as a therapeutic strategy in PC. Our studies clearly prove the fact that combining CAIX inhibitors with SOC chemotherapy agents demonstrates significantly more efficacy than single agents alone [68,69]. Furthermore, considering the significance of CAIX in acidosis of TME several studies employing anti-CAIX monoclonal antibodies against BC, CRC, CML, GC, and NSCLC are ongoing [66]. Several clinical phase studies have also been conducted using anti-CAIX monoclonal antibodies against RCC [66]. Importantly, the first clinical study to validate the safety of a first generation CD4TM - CAR containing scFv has been developed based on the murine antibody (anti-CAIX G250) expressed on the surface of primary T cells [66]. More recently, scientists have compared in pre-clinical setting the second-generation (BBζ and 28ζ) with third generation (28BBζ) carbonic anhydrase IX targeted CAR constructs in clear-cell renal cell carcinoma (ccRCC) in SK-RC-59 cell bearing NSG-SGM3 mouse model. Their findings confirmed that BBζ CAR4/8 cells are a highly potent and could be clinically applied for ccRCC [70].

Cellular and non-cellular components in tumor microenvironment (TME): TME is composed of various cellular and non-cellular components, including cancer-associated fibroblasts (CAFs), immune cells, extracellular matrix (ECM), blood vessels, signaling molecules each contributing to the intrinsic resistance in PC. Hypoxia induced TME in PC is characterized by dense stroma and immune cell infiltration. The stroma acts as a physical barrier, limiting drug delivery to cancer cells. PC-associated CAFs are a key component of the stroma and contribute to therapy resistance by secreting ECM proteins such as collagen and fibronectin that impede drug penetration, secrete pro-tumorigenic growth factors e.g., TGF-β, IL-6, and CXCL12 that promote tumor cell survival, proliferation, and resistance to apoptosis [71]. The emerging evidence highlights the critical role of MDSCs in driving resistance mechanisms in PC. As a part of immune escape strategies, tumor cells actively recruit MDSCs to suppress T cell activation. These cells secrete immunosuppressive molecules, including arginase, nitric oxide, and ROS to establish an immune-privileged microenvironment that supports PC cell survival. Moreover, in hypoxic tumor regions MDSCs produce pro-angiogenic factors such as VEGF, bFGF, and MMPs facilitating neovascularization and promoting tumor growth. Their role extends to extracellular matrix (ECM) remodeling, enabling PC cells to invade surrounding tissues. Additionally, MDSCs shield PC stem cells from cytotoxic treatments influenced by tumor-secreted factors like GM-CSF, G-CSF, IL-6, and IL-1β which drive MDSC recruitment and differentiation from bone marrow progenitors. HIFs further amplify MDSC expansion and their immunosuppressive functions within hypoxic tumor regions. MDSCs interact with other immune cells such as macrophages, skewing them toward the immunosuppressive M2 phenotype while impairing dendritic cell maturation and reducing antigen presentation to T cells. Given their role as key drivers of resistance to therapies in PC, targeting MDSCs has emerged as a promising therapeutic strategy. These approaches include inhibiting MDSC recruitment or expansion by blocking GM-CSF, G-CSF, or CXCR2 signaling pathways and such strategies are under pre-clinical/discovery phase [72].

ECM components such as hyaluronan and fibronectin interact with cell surface receptors (e.g., integrins) on cancer cells, activating signaling pathways that promote cell survival, migration, and resistance to apoptosis. Interestingly, CSCs interact with hyaluronan, activating PI3K/Akt and Ras/MAPK signaling pathways that confer resistance to apoptosis and promote cell survival [73]. CSCs via expression of markers such as CD44, ALDH1, CD133, EpCAM, and DCLK1 play pivotal roles in the intrinsic resistance of PC by enhancing CSCs' self-renewal, quiescence, apoptosis resistance, drug efflux capabilities, and interaction with survival pathways. Many CSCs are in a dormant or quiescent state, characterized by low proliferation rates allowing them to evade therapies that target dividing cells, such as chemotherapy and radiotherapy. Quiescent CSCs can later re-enter the cell cycle and regenerate the tumor [74]. These cells have higher expression levels of ABCG2, drug efflux transporters which pump out chemotherapeutic drugs, reducing their intracellular levels and effectiveness. Furthermore, activation of the Wnt/β-catenin pathway is closely associated with CSC and EMT properties and promotes resistance to apoptosis and enhances the self-renewal capacity of CSCs. EMT is regulated by several key signaling pathways, each contributing to therapy resistance in PC. TGF-β signaling is a major inducer of EMT that promotes expression of Snail, SMAD7 and Zeb1 which inhibits apoptosis and promotes cell survival [75]. Snail represses E-cadherin expression, a hallmark of EMT, leading to decreased cell adhesion and increased migratory capacity. Snail is also involved in the upregulation of anti-apoptotic proteins thereby enhancing resistance to apoptosis induced by chemotherapy [76]. Like Snail, Slug represses E-cadherin and promotes mesenchymal characteristics. Slug also induces resistance to DNA damage-induced apoptosis through the activation of the DNA repair enzyme PARP1 [77]. Additionally, Zeb1 also interacts with microRNAs, particularly the miR-200 family, which are known to suppress EMT. By downregulating these microRNAs, Zeb1 promotes a mesenchymal and drug-resistant phenotype [78].

Hypoxia induced metabolic reprogramming in PC: Metabolic reprogramming in PC is a hallmark of cancer cells contributing significantly to intrinsic resistance. It involves alterations in glucose and glutamine metabolism, lipid synthesis, redox homeostasis, and mitochondrial dynamics [79]. These adaptations support rapid proliferation and contribute to the overall resistance phenotype [80]. Glutamine is another crucial nutrient rewired in PC cells. The cells rely heavily on glutamine for anaplerosis and as a source of energy and biomass. Glutamine fuels TCA cycle and provides nitrogen for nucleotide and protein synthesis, thereby supporting rapid proliferation and resistance to metabolic stress induced by chemotherapy [81]. Altered lipid metabolism in PC cells increases de novo fatty acid synthesis, supports membrane biogenesis, and provides a source of energy for rapid proliferation, resistance to apoptosis and chemotherapy-induced stress [82]. PC cells maintain altered redox homeostasis due to enhanced expression of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) that helps PC cells neutralize ROS, thereby reducing oxidative damage and increasing resistance to chemotherapy [83]. Mitochondrial metabolism also plays a dual role in PC, supporting both energy production and cell survival under stress conditions.

Extrinsic resistance in PC: Extrinsic factors also cause resistance in PC apart from the intrinsic factors. They involve a complex interplay of mechanisms that hinder effective drug delivery and promote resistance to therapies.

Adaptive genetic changes/secondary genetic mutations in PC: Cancer cells with acquired BRCA2 reversion mutations that restore the function of BRCA2, which was initially mutated, leads to resistance to drugs like PARP inhibitors. The development of these secondary reversion mutations are possible mechanisms of resistance in BRCA2-mutant PDAC [84]. Additionally, mutations that activate the NRF2 pathway can enhance antioxidant defenses and confer resistance to oxidative stress-inducing chemotherapies in PC.

Drug transport, delivery, and metabolism: The overexpression of MDR1 and ABC transporters ABCG2 (BCRP) and ABCC1 (MRP1), altered expression of metabolic genes (GST and CYP450) involved in drug efflux, and physical barriers within TME actively pump various chemotherapeutic drugs out of cancer cells, reducing intracellular drug concentrations. MDR1 contributes significantly to resistance in PC against a broad spectrum of chemotherapy drugs, including Gemcitabine and Paclitaxel [85]. Upregulation of metabolic enzymes such as GSTs and CYPs enhances drug metabolism and detoxification. These enzymes catalyze the conjugation and degradation of chemotherapy drugs, reducing their efficacy and contributing to resistance in PC [86].

Immune evasion: PC is particularly resistant to treatment, partly due to its ability to evade the immune system which allows tumor growth without being detected and destroyed by the body's immune defenses. Tumor cells often downregulate MHC class I, which present antigenic peptides to CTLs and impairs the ability of T cells to recognize and target tumor cells. The upregulation of anti-apoptotic factors by tumor cells inhibits apoptosis making them more resistant to killing by CTLs and NK cells. The production of high levels of metabolic by-products like adenosine and lactic acid, suppress immune cell function and promote a more tolerant environment. The dense ECM in PC acts as a physical barrier, impeding the infiltration of immune cells into tumors. The immune evasion in PC contributes to extrinsic resistance in several ways [71].

Inflammation: Inflammation plays a critical role in the development and progression of many cancers, including PC. Chronic inflammation contributes to tumor growth, metastasis, and resistance to therapy due to the involvement of various inflammatory pathways and cellular components that create a supportive niche for cancer cells. Inflammatory cells act as a mediator within the TME interacting with cancer cells and other stromal components, contributing to a pro-tumorigenic milieu. NF-κB is activated by various stimuli, including cytokines (e.g., TNF-α, IL-1β), stress signals, and microbial infections. The constitutive activation of NF-κB regulates genes involved in cell survival, proliferation, angiogenesis, and inflammation and simultaneously promote the production of pro-inflammatory cytokines (e.g., IL-6, IL-8), anti-apoptotic proteins, and growth factors [87]. Also, STAT3 is frequently activated by IL-6 and EGF through the JAK pathway and promotes tumor cell proliferation, survival, angiogenesis, and immune evasion by regulating the expression of genes involved in these processes [88]. Additionally, overexpression of COX-2 and PEG2 pathway by inflammatory stimuli and oncogenic signals promote tumor growth, invasion, and angiogenesis and modulates the immune response, favoring a more immunosuppressive environment [89].

Microbiome in PC: The human microbiome, particularly the gut microbiome, has emerged as a significant factor influencing cancer progression and response to therapy. The microbiome in PC can originate from gut via translocation through the bloodstream or bile duct and from the oral cavity through the duodenum. Recent studies have shown that PC tumors harbor distinct microbial communities, different from those in healthy pancreatic tissue [90]. The complex interplay of microbial communities directly impacts tumor biology, immune modulation, and interactions with therapeutic agents. Further, these microbial communities interact with host cells and influence disease processes including TME [91]. The microbiome can trigger chronic inflammation within the pancreas due to inflammatory cytokine produced by pathogenic bacteria leading to tumor progression and resistance. Certain microbial communities may further create an immunosuppressive TME through the recruitment of Tregs, MDSCs, and the production of anti-inflammatory cytokines. Moreover, microbes can modulate the expression of immune checkpoint molecules like PD-L1 on tumor cells, which inhibit the immune response. Microbial components such as lipopolysaccharides (LPS) can activate Toll-like receptors (TLRs) on immune cells promoting tumor cell survival, inflammation, and resistance to apoptosis [92,93]. Some bacteria produce toxins or secondary bile acids that can directly damage DNA or promote cellular changes that lead to tumorigenesis. Fusobacterium nucleatum has been found in PC tissues and is associated with poor prognosis as it can inhibit immune response and promote chemoresistance through modulation of autophagy and activation of oncogenic pathways [94]. Overall, microbiome in PC can affect the efficacy of treatments, contributing to extrinsic resistance. Thus, identifying specific microbial signatures associated with resistance can serve as biomarkers to predict treatment responses and guide therapy decisions [95].

Considering the various factors, intrinsic and extrinsic leading to therapy evasion mechanisms, numerous strategies can be envisioned to circumvent resistance. Clinical trials utilizing innovative combinatorial chemotherapy regimens have shown encouraging improvements in survival rates. These advancements in treatment options have greatly broadened even for patients with locally advanced and metastatic disease. Recently, ONIVYDE (irinotecan liposome injection) used in combination with oxaliplatin, fluorouracil, and leucovorin (NALIRIFOX) received an approval as a first-line therapy of PDAC based on better OS and PFS compared with Gem + NabP [96]. Similarly, combinatorial strategies to overcome efflux transporter (P-gp, MRPs and BCRP) mediated resistance has also been considered as an encouraging approach [97].

PDAC treatment resistance has also been attributed to its ability to evade the immune system often due to downregulation of MHC class I, which presents antigenic peptides to CTLs and impairs the ability of T cells to recognize and target tumor cells. Importantly, tumor cells express immune checkpoint proteins (PD-L1 or L2) binding to PD-1 receptor on T cells to diminish T cell activation and induce T cell exhaustion, leading to reduced immune responses against the tumor. CTLA-4 is another checkpoint protein that downregulates immune responses. Considering this, drugs targeting PD-1/PD-L1 or CTLA-4 pathways which could reactivate T cells and restore anti-tumor immune responses. However, further research is required to comprehend the failure of checkpoint inhibitors [98].

Vaccines against PC is another upcoming strategy specifically targeting KRAS for better prognosis in people with high-risk. Additionally, strategies to inhibit KRAS also include direct KRAS-targeted therapies, modulation of upstream and downstream signaling, KRAS-specific siRNA, and novel combination therapies integrating KRAS inhibitors with immune checkpoint blockade, PARP inhibitors, CDK4/6 inhibitors, and autophagy modulators [99].

Claudin 18.2 is an additional promising marker highly expressed in PDAC and corelates with initiation and progression of disease. PT886, an IgG1 bispecific antibody targeting CLDN18. 2, and CD47 is in clinical trial. It mediates antibody-dependent cellular cytotoxicity (ADCC) and enhances antibody-dependent cellular phagocytosis (ADCP), stimulating the innate immune system and subsequently increasing tumor neoantigen presentation and T cell-mediated cancer cell killing [100]. Additionally, treatment with oncolytic adenoviruses (OV) given intravenously would present a better approach for improving clinical outcomes for PDAC patients. It is envisaged that OV based gene therapy would be a game changer due to the advent of next generation OVs associated with better specificity and selectivity when offered in combination with personalized treatment regimens customized with the help of AI. OV (VCN-01) in combination with chemotherapy is being assessed in the VIRAGE clinical trial for PC. VCN-01 replicates in tumor cells and disrupts the stroma, potentially allowing the treatment to be more effective [101].

Additionally, strategies employing adoptive cell therapy that uses CAR-T cells to better recognize and attack tumor cells, synergistic combination of immunotherapy, chemotherapy, and agents targeting the TME may lead to better prognosis [102].

PDAC remains one of the deadliest cancers as the incidence and mortality continue to rise. Importantly, it has been forecast that PC will surpass breast, prostate, and colorectal cancers as the leading cause of cancer-related death in the US by the year 2030. Early detection is crucial, particularly for high-risk groups who could benefit from targeted screening programs. PDAC at initial stage is generally less heterogeneous, exhibits limited stromal proliferation, better vascularization which otherwise would impede drug delivery causing resistance. Notably, as PDAC progresses, tumors accumulate additional genetic mutations and epigenetic alterations and often create an immunosuppressive TME that leads to resistance and diminishes the efficacy of immune-based therapies. Therefore, early diagnosis is of paramount importance which may provide a safe window to address various resistance mechanisms, thereby improving the effectiveness of therapeutic interventions. Ongoing research into detection methods (liquid biopsy using CTC, ctDNA, cfDNA or exosomes) including the development of novel biomarkers (serological signatures, autoantibodies, epigenetic markers, and miRNAs) and advanced imaging techniques, holds promise for enhancing patient outcomes. While progress on the diagnosis front has been conscientiously measured, it is important to understand the numerous causes of PDAC therapy resistance. This review specifically focuses on the intrinsic and extrinsic mechanisms exemplified by a complex microenvironment that along with numerous metabolic changes support several interactions leading upto therapeutic escape mechanisms (Figure 4). To address these mechanisms, it is important that efforts are directed towards robust and evidence-based research on cancer therapy. Significant progress has been achieved in understanding the molecular mechanisms of PC. Notably, artificial intelligence (AI) is becoming a transformative tool in areas such as medical imaging, biomarker identification, genetic research, and treatment strategy development. We do believe that with the continuous innovation and development of various novel biotechnology, integrated development of biology, diagnostic, pharmacy, medicines and other disciplines, breakthroughs will be achieved in treating PC to benefit a greater number of patients.

This paper was not funded.

Authors have no conflict of interest to share.

Authors are thankful to Cipla Ltd for supporting the scope of the manuscript.