Renal cell carcinoma (RCC) is a common cancer of the genitourinary tract that has very poor survival outcomes if metastatic. RCC is now understood to be composed of several different types of cancer with different genetic features and varied clinical responses. Histological diagnosis has been the primary method to diagnose RCC and has been used to define three major RCC subtypes, including the most common subtype, clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (PRCC; further divided into two subtypes), and chromophobe renal cell carcinoma (ChRCC)^[1]. More recent comparative genomic and phenotypic analysis has identified mutations and epigenetic modifications associated with different histological subtypes^[2]. Across all subtypes, increased DNA hypermethylation and gene alterations in CDKN2A were associated with a poor prognosis as was an increased Th2 immune gene signature. For ccRCC, increased levels of mRNA transcripts associated with ribose metabolism and the immune response were associated with poor survival. ccRCC is also defined by the early loss of chromosome 3p, which in turn causes a loss of heterozygosity for the VHL, PBRM1, SETD2, and BAP1 tumor suppressor genes and subsequent mutation of these genes that leads to tumorigenesis^[3]. There is also a subset of ChRCC with a unique metabolic expression pattern that is associated with extremely poor survival^[2]. PRCC can be classified as type 1, for which PBRM1 mutations are linked to poor survival but type 2 PRCC has increased expression of glycolysis and metabolism-related mRNA transcripts^[2].

ccRCC tumors with VHL mutations show overexpression of vascular endothelial growth
factor (VEGF) and hypoxia-inducible factors (HIFs) can contribute to angiogenesis and cancer progression. Similarly, some RCCs show hyperactivation of the serine/threonine kinase mammalian target of rapamycin (mTOR), which can lead to the overproduction of VEGF. VEGFR inhibitors and anti-VEGF antibodies have been tested as therapies for RCC, and the anti-VEGF antibody bevacizumab has been approved for use in combination with IFN-α for metastatic RCC^[4]. The mTOR inhibitors everolimus and temsirolimus have also been approved for the treatment of RCC, typically in combination with tyrosine kinase inhibitors (TKIs)^[5]. Combination therapies that target VEGF and mTOR are considered more effective since they work in concert to target tumor growth and vascularization, whereas sequential treatments are typically associated with a greater likelihood of tumors developing resistance^[6]. Unfortunately, these combination therapies are associated with undesirable toxicities and have not been linked with durable responses^[7].

Advances in immunotherapy have led to transformative treatment options for RCC. Immune checkpoint blockade (ICB) has been one promising treatment strategy, and the FDA approved the use of anti-PD-1/PD-L1 (nivolumab) for the treatment of advanced ccRCC in 2015^[8]. A follow-up study showed that anti-PD-1 was associated with the best clinical benefit in ccRCC carrying loss-of-function mutations in PBRM1, which appears to affect tumor expression profiles in such a way to maintain responsiveness to checkpoint blockade^[9]. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is another checkpoint molecule targeted for checkpoint blockade with the monoclonal antibody ipilimumab. Similar to PD-L1 blockade, CTLA-4 blockade was associated with a partial response against metastatic RCC. Combination therapies have shown much more durable responses in patients with advanced RCC, and the FDA approved the use of nivolumab plus ipilimumab for the treatment of intermediate or high-risk metastatic RCC in 2018^[10]. ICB blockade in combination with TKI are also under investigation as alternative first or second-line treatments. Unfortunately, PD-1/PD-L1 blockade has been less successful for PRCC^[11] and ChRCC^[12], and further studies are needed to identify therapeutic targets in these forms of RCC. Clinical studies with other checkpoint blockade targets are currently underway and are likely to provide new treatment options to RCC patients^[13].

The future of RCC therapies relies on the identification of new molecules or pathways in tumor cells that can be targeted therapeutically without causing toxicity or promoting resistance. Advances in single-cell omics are leading the way in terms of target identification and understanding how different forms of RCC progress.

^[1] Hsieh JJ, Purdue MP, Signoretti S, Swanton C, Albiges L, Schmidinger M, Heng DY, Larkin J, Ficarra V. Renal cell carcinoma. Nat Rev Dis Primers. 2017;3:17009.

^[2] Ricketts CJ, De Cubas AA, Fan H, et al. The Cancer Genome Atlas Comprehensive Molecular Characterization of Renal Cell Carcinoma [published correction appears in Cell Rep. 2018 Jun 19;23(12):3698]. Cell Rep. 2018;23(1):313-326.e5.

^[3] Turajlic S, Swanton C, Boshoff C. Kidney cancer: The next decade. J Exp Med. 2018;215(10):2477-2479. doi:10.1084/jem.20181617

^[4]Rini BI, Halabi S, Rosenberg JE, et al. Phase III trial of bevacizumab plus interferon alfa versus interferon alfa monotherapy in patients with metastatic renal cell carcinoma: final results of CALGB 90206. J Clin Oncol. 2010;28(13):2137-2143.

^[5] Leonetti A, Leonardi F, Bersanelli M, Buti S. Clinical use of lenvatinib in combination with everolimus for the treatment of advanced renal cell carcinoma. Ther Clin Risk Manag. 2017 Jun 30;13:799-806.

^[6] Gore ME, Larkin JM. Challenges and opportunities for converting renal cell carcinoma into a chronic disease with targeted therapies. Br J Cancer. 2011 Feb 1;104(3):399-406.

^[7] Escudier B: Emerging immunotherapies for renal cell carcinoma. Ann Oncol 23:viii35-viii40, 2012 (suppl 8).

^[8] Motzer RJ, Escudier B, McDermott DF, et al. Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med. 2015;373(19):1803-1813.

^[9] Miao D, Margolis CA, Gao W, Voss MH, Li W, Martini DJ, Norton C, Bossé D, Wankowicz SM, Cullen D, Horak C, Wind-Rotolo M, Tracy A, Giannakis M, Hodi FS, Drake CG, Ball MW, Allaf ME, Snyder A, Hellmann MD, Ho T, Motzer RJ, Signoretti S, Kaelin WG Jr, Choueiri TK, Van Allen EM. Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma. Science. 2018 Feb 16;359(6377):801-806.

^[10] Hammers HJ, Plimack ER, Infante JR, Rini BI, McDermott DF, Lewis LD, Voss MH, Sharma P, Pal SK, Razak ARA, Kollmannsberger C, Heng DYC, Spratlin J, McHenry MB, Amin A. Safety and Efficacy of Nivolumab in Combination With Ipilimumab in Metastatic Renal Cell Carcinoma: The CheckMate 016 Study. J Clin Oncol. 2017 Dec 1;35(34):3851-3858.

^[11] de Vries-Brilland, Manon, et al. Are immune checkpoint inhibitors a valid option for papillary renal cell carcinoma? A multicentre retrospective study. European Journal of Cancer 2020; 136:76-83.

^[12] Schwartzman, William, et al. "Safety and efficacy of immune checkpoint inhibitors (ICI) in metastatic non-clear cell renal cell carcinoma (nccRCC): An institutional experience." J. Clin Oncol. 2020: 640.

^[13] Mollica V, Di Nunno V, Gatto L, Santoni M, Cimadamore A, Cheng L, Lopez-Beltran A, Montironi R, Pisconti S, Battelli N, Massari F. Novel Therapeutic Approaches and Targets Currently Under Evaluation for Renal Cell Carcinoma: Waiting for the Revolution. Clin Drug Investig. 2019 Jun;39(6):503-519.

Tumor-infiltrating lymphocyte (TIL)-based immunotherapy is currently at the forefront of cutting-edge immuno-oncology treatments. TILs are a type of adoptive cellular therapy (ACT) using lymphocytes that are found within tumor tissues; most of these lymphocytes are T cells that can specifically target tumor cells. For TIL-based therapies, these T cells are harvested from a tumor biopsy, expanded ex vivo, and infused back into the patient. Advances in TIL-based therapies are driven by preclinical characterization and screening of TILs against a wide array of tumor types.

Consider these factors related to preclinical TIL therapy studies as you develop new research protocols:

1. Wanted Alive, Not Dead: The development of plate-based TIL assays offers users flexibility and scalability, but it is critical to assess the viability of cultured TILs at the outset of any of these assays. Some TIL cultures are prone to high levels of cell death over time, and culture conditions may need to be optimized to include different cytokines and growth factors to promote viability as well as growth and expansion of tumor-specific T cells.
   
   
2.  Cell Selection: Optimal TIL culture conditions should enhance the expansion of tumor-specific T cells but may also involve a step to deplete other cells prior to expansion. This may involve the depletion of adherent cell subsets with immunosuppressive characteristics such as myeloid-derived suppressor cells (MDSCs). Removing these cells from ex vivo culture allows TILs to expand and differentiate into T cells with tumor-specific cytotoxic activity.
   
   
3. TIL Test Drive: Ex vivo expansion of TILs must include functional assays in order to ascertain if these cells have anti-tumor properties. Analysis typically includes flow cytometry-based immunophenotyping and characterization of cytokine production and tumor-specific cytotoxicity. An ideal TIL product will show tumor-specific activity with limited off-target effects, and TILs will retain a phenotype that is predicted to be well-tolerated in a patient upon re-infusion.

Preclinical TIL studies are continuing to expand the usage of TIL-based therapies against a wide range of solid tumors, and this field of study will advance further as ex vivo culture and analysis techniques improve.

Mouse models have been the workhorses of preclinical immuno-oncology (IO) research, and advances in mouse model development have expanded to applications for nearly all types of solid tumors and hematological malignancies. Preclinical evaluation of experimental immunotherapies has been advanced by syngeneic and humanized mouse models. Syngeneic mice are one of the most established types of models used in cancer research, whereas humanized mice are a contemporary mouse model that has been critical to the screening of immunotherapeutic agents. Here we highlight features of syngeneic and humanized mouse models and define which models are most relevant to different phases of preclinical IO research.

Syngeneic Tumor Models

Syngeneic tumor models are created by transplantation of tumor cell lines into immunocompetent mice with the same genetic background as the cell line^[1]. Tumors can be transplanted intravenously or subcutaneously into mice and typically grow rapidly over several weeks. Different types of tumor cell lines can be used in this type of model, including spontaneous, transgenic, or carcinogen-induced tumor cell lines. Syngeneic mouse models are best suited for screening novel IO agents or gaining insight into anti-tumor responses in the context of an intact immune system. Given the rapid growth of tumors in syngeneic mice, these models are less well suited to studying early events in tumor growth associated with cancer stem cells or understanding the contributions of heterogeneous tumor microenvironments, and these models typically do not recapitulate the mutational heterogeneity observed in human tumors^[2].

Humanized Tumor Models

Humanized tumor models are a more recent addition to preclinical IO research that provide valuable insight into how individual tumors from patients (xenografts) respond to experimental therapies. Prior to the development of humanized mouse models, human xenograft models were used for screening cytotoxic or immunotherapeutic agents like chimeric antigen receptor (CAR) T cells^[3], and these models use human tumor cell lines or patient-derived specimens transplanted into immunocompromised host mice. Different immunocompromised models can be used, including athymic mice that lack T cells or severe combined immunodeficiency (SCID) models that lack all adaptive immune responses. Humanized mice have been engineered from immunocompromised mouse strains that include genetic mutations in other adaptive immune functions that allow for engraftment of human hematopoietic cells. The NOD/SCID IL2rγ chain knockout (NSG) mouse (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) is one of the most used combined immunodeficiency models that can be engrafted with human hematopoietic cells and primary human tumors^[4]. These patient-derived xenograft (PDX) models are useful for evaluated experimental IO therapies in the context of the human immune system and can use human immune cells from the same or different donor as the tumor source. PDX models are suited to evaluating experimental therapies in the context of a genetically heterogeneous tumor and better recapitulates aspects of the tumor microenvironment. Tumors can be grafted either orthotopically or subcutaneously and this also impacts how tumors grow and respond to experimental treatments^[5]. Given the heavily modified nature of the NSG immune system, these models do not always reflect responses observed in humans during clinical trials. Nonetheless, NSG mice and similarly modified humanized mice offer valuable insights into the efficacy of IO candidates.

Mouse models are constantly being refined and improved to better reflect human physiology. Both syngeneic and humanized mouse models serve as valuable tools to preclinical IO research and accelerate the screening and evaluation of novel therapeutics.

^[1] Olson B, Li Y, Lin Y, Liu ET, Patnaik A. 2018. Mouse Models for Cancer Immunotherapy Research. Cancer Discov. 8(11):1358-1365.

^[2] Hanahan D, Weinberg RA. 2011. Hallmarks of cancer: the next generation. Cell. 144:646–74.

^[3] Siegler EL, Wang P. (2018). Preclinical models in chimeric antigen receptor–engineered T-cell therapy. Hum. Gene Ther., 29(5), 534-546.

^[4] Okada S, Vaeteewoottacharn K, Kariya R. 2019. Application of Highly Immunocompromised Mice for the Establishment of Patient-Derived Xenograft (PDX) Models. Cells. 8(8):889.

^[5] Byrne AT, Alférez DG, Amant F, Annibali D, Arribas J, Biankin AV, Bruna A, Budinská E, Caldas C, Chang DK, Clarke RB. 2017. Interrogating open issues in cancer precision medicine with patient-derived xenografts. Nature Reviews Cancer. 17(4):254.

Renal cell carcinoma (RCC) is a common cancer of the genitourinary tract that has very poor survival outcomes if metastatic. RCC is now understood to be composed of several different types of cancer with different genetic features and varied clinical responses. Histological diagnosis has been the primary method to diagnose RCC and has been used to define three major RCC subtypes, including the most common subtype, clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (PRCC; further divided into two subtypes), and chromophobe renal cell carcinoma (ChRCC)^[1]. More recent comparative genomic and phenotypic analysis has identified mutations and epigenetic modifications associated with different histological subtypes^[2]. Across all subtypes, increased DNA hypermethylation and gene alterations in CDKN2A were associated with a poor prognosis as was an increased Th2 immune gene signature. For ccRCC, increased levels of mRNA transcripts associated with ribose metabolism and the immune response were associated with poor survival. ccRCC is also defined by the early loss of chromosome 3p, which in turn causes a loss of heterozygosity for the VHL, PBRM1, SETD2, and BAP1 tumor suppressor genes and subsequent mutation of these genes that leads to tumorigenesis^[3]. There is also a subset of ChRCC with a unique metabolic expression pattern that is associated with extremely poor survival². PRCC can be classified as type 1, for which PBRM1 mutations are linked to poor survival but type 2 PRCC has increased expression of glycolysis and metabolism related mRNA transcripts².

ccRCC tumors with VHL mutations show overexpression of vascular endothelial growth
factor (VEGF) and hypoxia-inducible factors (HIFs), which can contribute to angiogenesis and cancer progression. Similarly, some RCC show hyperactivation of the serine/threonine kinase mammalian target of rapamycin (mTOR), which can lead to overproduction of VEGF. VEGFR inhibitors and anti-VEGF antibodies have been tested as therapies for RCC, and the anti-VEGF antibody bevacizumab has been approved for use in combination with IFN-α for metastatic RCC^[4]. The mTOR inhibitors everolimus and temsirolimus have also been approved for the treatment of RCC, typically in combination with tyrosine kinase inhibitors (TKIs)^[5]. Combination therapies that target VEGF and mTOR are considered more effective since they work in concert to target tumor growth and vascularization, whereas sequential treatments are typically associated with a greater likelihood of tumors developing resistance^[6]. Unfortunately, these combination therapies are associated with undesirable toxicities and have not been linked with durable responses^[7].

Advances in immunotherapy have led to transformative treatment options for RCC. Immune checkpoint blockade (ICB) has been one promising treatment strategy, and the FDA approved the use of anti-PD-1/PD-L1 (nivolumab) for the treatment of advanced ccRCC in 2015^[8]. A follow up study showed that anti-PD-1 was associated with the best clinical benefit in ccRCC carrying loss-of-function mutations in PBRM1, which appears to affect tumor expression profiles in such a way to maintain responsiveness to checkpoint blockade^[9]. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is another checkpoint molecule that has been targeted for checkpoint blockade with the monoclonal antibody ipilimumab. Similar to PD-L1 blockade, CTLA-4 blockade was associated with a partial response against metastatic RCC. Combination therapies have shown much more durable responses in patients with advanced RCC, and the FDA approved the use of nivolumab plus ipilimumab for the treatment of intermediate or high risk metastatic RCC in 2018^[10]. ICB blockade in combination with TKI are also under investigation as alternative first or second line treatments. Unfortunately, PD-1/PD-L1 blockade has been less successful for PRCC^[11] and ChRCC^[12], and further studies are needed to identify therapeutic targets in these forms of RCC. Clinical studies with other checkpoint blockade targets are currently underway and are likely to provide new treatment options to RCC patients^[13].

The future of RCC therapies relies on the identification of new molecules or pathways in tumor cells that can be targeted therapeutically without causing toxicity or promoting resistance. Advances in single-cell omics are leading the way in terms of target identification and understanding how different forms of RCC progress.

^[1] Hsieh JJ, Purdue MP, Signoretti S, Swanton C, Albiges L, Schmidinger M, Heng DY, Larkin J, Ficarra V. Renal cell carcinoma. Nat Rev Dis Primers. 2017;3:17009.

^[2] Ricketts CJ, De Cubas AA, Fan H, et al. The Cancer Genome Atlas Comprehensive Molecular Characterization of Renal Cell Carcinoma [published correction appears in Cell Rep. 2018 Jun 19;23(12):3698]. Cell Rep. 2018;23(1):313-326.e5.

^[3] Turajlic S, Swanton C, Boshoff C. Kidney cancer: The next decade. J Exp Med. 2018;215(10):2477-2479. doi:10.1084/jem.20181617

^[4]Rini BI, Halabi S, Rosenberg JE, et al. Phase III trial of bevacizumab plus interferon alfa versus interferon alfa monotherapy in patients with metastatic renal cell carcinoma: final results of CALGB 90206. J Clin Oncol. 2010;28(13):2137-2143.

^[5] Leonetti A, Leonardi F, Bersanelli M, Buti S. Clinical use of lenvatinib in combination with everolimus for the treatment of advanced renal cell carcinoma. Ther Clin Risk Manag. 2017 Jun 30;13:799-806.

^[6] Gore ME, Larkin JM. Challenges and opportunities for converting renal cell carcinoma into a chronic disease with targeted therapies. Br J Cancer. 2011 Feb 1;104(3):399-406.

^[7] Escudier B: Emerging immunotherapies for renal cell carcinoma. Ann Oncol 23:viii35-viii40, 2012 (suppl 8).

^[8] Motzer RJ, Escudier B, McDermott DF, et al. Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N Engl J Med. 2015;373(19):1803-1813.

^[9] Miao D, Margolis CA, Gao W, Voss MH, Li W, Martini DJ, Norton C, Bossé D, Wankowicz SM, Cullen D, Horak C, Wind-Rotolo M, Tracy A, Giannakis M, Hodi FS, Drake CG, Ball MW, Allaf ME, Snyder A, Hellmann MD, Ho T, Motzer RJ, Signoretti S, Kaelin WG Jr, Choueiri TK, Van Allen EM. Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma. Science. 2018 Feb 16;359(6377):801-806.

^[10] Hammers HJ, Plimack ER, Infante JR, Rini BI, McDermott DF, Lewis LD, Voss MH, Sharma P, Pal SK, Razak ARA, Kollmannsberger C, Heng DYC, Spratlin J, McHenry MB, Amin A. Safety and Efficacy of Nivolumab in Combination With Ipilimumab in Metastatic Renal Cell Carcinoma: The CheckMate 016 Study. J Clin Oncol. 2017 Dec 1;35(34):3851-3858.

^[11] de Vries-Brilland, Manon, et al. Are immune checkpoint inhibitors a valid option for papillary renal cell carcinoma? A multicentre retrospective study. European Journal of Cancer 2020; 136:76-83.

^[12] Schwartzman, William, et al. "Safety and efficacy of immune checkpoint inhibitors (ICI) in metastatic non-clear cell renal cell carcinoma (nccRCC): An institutional experience." J. Clin Oncol. 2020: 640.

^[13] Mollica V, Di Nunno V, Gatto L, Santoni M, Cimadamore A, Cheng L, Lopez-Beltran A, Montironi R, Pisconti S, Battelli N, Massari F. Novel Therapeutic Approaches and Targets Currently Under Evaluation for Renal Cell Carcinoma: Waiting for the Revolution. Clin Drug Investig. 2019 Jun;39(6):503-519.