In the manuscript titled Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model, our research group generated a novel PDX model TU-BcX-2K1 (2K1 for short) by implanting a piece of primary patient tumor derived from a pre-neoadjuvant 59-year-old Black woman with invasive ductal carcinoma, triple negative subtype, into SCID/Beige mice [1]. This report is relevant because disparities in breast cancer statistics have been noted, with Black women developing the disease at a younger age, having higher incidence rates, and having worse outcomes compared to White women [2,3]. Furthermore, 2K1 is derived from a Triple Negative Breast Cancer (TNBC) tumor, a subtype of breast cancer that is more aggressive, metastatic, resistant to chemotherapeutics, and has less treatment options compared to others. By generating and characterizing a novel TNBC PDX model, we hope to provide an invaluable tool for the community of breast cancer researchers and contribute to the growing library of similar models. This commentary will focus on the two important aspects of cancer research brought up by this manuscript: cancer models and biological racial differences and disparities.

Immortalized cell lines have been the gold standard model for research since the creation of HeLa (named after the patient, Henrietta Lacks), a famous cell line derived from cervical cancer. Cell lines are a powerful tool for research because they divide indefinitely, are easy to maintain, and are composed of a single clonal population, thus reducing variability between experiments. In addition to cervical cancer research, HeLa cells have contributed to other fields of research such as virology (used to test the first polio vaccine), genetics (used to identify 23 pairs of human chromosomes), and even space microbiology (cells sent to space on Sputnik-6) [4]. Cell lines also have their limitations. For example, most therapies fail in phase 3 clinical trials despite promising in-vitro results with cell lines [5]. Potential explanations for why this happens include misidentification/mischaracterization of cell lines, biological variability between patients, genetic drift, and failure of the cell lines to recapitulate the complex interactions found within a disease microenvironment. Due to these limitations of cell lines in translational research, additional models need to be developed to advance science.

Patient Derived Xenografts (PDX’s) are a more complex model that is generated by growing tumors in a mammalian host system (typically mice). Because the tumor is maintained and grown in a mammalian system, host cells can contribute to the complex microenvironment and sustain tumor growth similar to the original patient system. For example, PDX tumors develop blood vessels, maintain a tumor architecture, and can better mimic oxygen, nutrient, and hormone levels [6]. These qualities allow PDX’s to better predict clinical activity and successes of novel compounds for therapy [7]. In addition to using PDX models to test novel

compounds in-vivo, medium throughput compound screens can be performed by plating cells isolated from PDX tumors in 2D and 3D in-vitro conditions. Using this technique, we found PDX 2K1 to be sensitive to purine analogs, antimetabolites, and antitumor antibiotics while being resistant to alkylating agents in-vitro. Furthermore, we used 3D in-vitro culture conditions to screen for compound sensitivity. When compared to 2D, the 3D culture conditions resulted in higher drug resistance when treated at the same dose. This could be explained by the potential enrichment of cancer stem cells due to the non-adherent conditions. Nonetheless, we were able to identify imiquimod and ceritinib as effective against 2K1 in 3D conditions. These screens could rapidly be performed to provide personalized therapies in the clinical setting.

Although PDX models have been generally acknowledged for their strengths in providing a superior microenvironment compared to cell lines, there are limitations when it comes to studying the tumor immune microenvironment. Because PDX tumors are generated by implanting human derived tumors into a non-human mammalian host, immunocompromised animals must be used to prevent tissue rejection. In this manuscript, we utilized the SCID/Beige (CB17.Cg-Prkdc^scidLyst^bg-J/Crl) model from Charles River. This is a congenic mouse model that possesses the autosomal recessive mutations SCID and beige, resulting in severe combined immunodeficiency affecting B and T lymphocytes, and defective NK cells, respectively. Although this prevents host rejection of xenografted tissue, these mutations hinder studies aimed at the immunological components of the tumor microenvironment. With the advent of immunotherapy research, many groups have developed methods for generating a humanized immune system within mice. A commonly used technique involves sublethal irradiation of humanized mice followed by IV injection of CD34⁺ cord blood cells [8,9]. These cells then migrate to the host bone marrow and can differentiate into various cellular components of the immune system. Because this technique often leads to the eventual development of graft-vs.-host disease,

there is still room for improvement in terms of generating the “ideal” in-vivo model.

Despite cancer incidence and mortality rates steadily decreasing due to research efforts, there is a clear racial disparity in disease outcomes. For example, Black patients have the highest cancer death rates despite having similar incidence rates of breast cancer compared to White patients [10]. The cause for this phenomenon is most likely multifactorial with genetics, environmental, and socioeconomic influences playing a role. We would like to bring up the source of research samples as an often-overlooked source of disparities in oncological treatment. The PDXNet is a multi-institute collaboration initiated by the NCI to develop, characterize, and provide PDX data of different cancers to the research community to accelerate translational research. Despite being a consortium of 6 major research institutions, only 23 of the 334 PDX’s available were derived from Black patients; the majority of samples are from Caucasian patients  (https://portal.pdxnetwork.org/). Our intent is not to directly criticize these institutions but, rather, to provide an example of disparity in medical research. We believe that the PDXNet is already an impressive database and hope to emphasize the importance of more representative models to improve the accuracy of research results for diverse patient populations.

Research models, for the most part, have been historically derived from Caucasian patients. Although these models have greatly benefited the field of medical research, the biological differences associated with different races may have led to the clinical outcomes disparities that exist today. We hope to help close this gap by developing and characterizing a PDX model derived from a Black patient with TNBC for the research community. This characterization includes tumor growth rate, metastasis, histological observations, genetic analysis of common cancer associated pathways (EMT), cancer stem cells analysis, and a drug resistance/sensitivity panel. By developing a preclinical model derived from a Black patient, we hope to contribute to the research community by providing a valuable research tool that is able to address an aspect of cancer research disparity.

Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model is a manuscript detailing the development and characterization of novel PDX model 2K1. This model is generated by implanting tumor tissue from a 59-year-old black woman with TNBC into an immunodeficient mouse model. We hope to bring up the important topics of research models and research disparities in cancer research through this commentary. Because this model is maintained in a mammalian environment, it is a superior research model for studying the tumor microenvironment and tumor scaffold architecture in comparison to isolated 2D-cultured cell lines. Furthermore, this PDX model addresses the gap in research disparities because it is derived from a Black patient, a population that has higher risks and worse outcomes for breast cancers. In addition to PDX 2K1, our research group has also generated and characterized other PDX models that may be interesting to the research community [11-17]. Our research focus includes the tumor microenvironment, drug resistance, and cancer metastasis. Although this commentary only covers two components of the complex oncology research world, we hope that we were successful in bringing to light important aspects of research.