Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFß-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

The Tumor Microenvironment of Clear-Cell Ovarian Cancer.

Some patients with advanced clear-cell ovarian cancer (CCOC) respond to immunotherapy; however, little is known about the tumor microenvironment (TME) of this relatively rare disease. Here, we describe a comprehensive quantitative and topographical analysis of biopsies from 45 patients, 9 with Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stage I/II (early CCOC) and 36 with FIGO stage III/IV (advanced CCOC). We investigated 14 immune cell phenotype markers, PD-1 and ligands, and collagen structure and texture. We interrogated a microarray data set from a second cohort of 29 patients and compared the TMEs of ARID1A-wildtype (ARID1Awt) versus ARID1A-mutant (ARID1Amut) disease. We found significant variations in immune cell frequency and phenotype, checkpoint expression, and collagen matrix between the malignant cell area (MCA), leading edge (LE), and stroma. The MCA had the largest population of CD138+ plasma cells, the LE had more CD20+ B cells and T cells, whereas the stroma had more mast cells and αSMA+ fibroblasts. PD-L2 was expressed predominantly on malignant cells and was the dominant PD-1 ligand. Compared with early CCOC, advanced-stage disease had significantly more fibroblasts and a more complex collagen matrix, with microarray analysis indicating "TGFß remodeling of the extracellular matrix" as the most significantly enriched pathway. Data showed significant differences in immune cell populations, collagen matrix, and cytokine expression between ARID1Awt and ARID1Amut CCOC, which may reflect different paths of tumorigenesis and the relationship to endometriosis. Increased infiltration of CD8+ T cells within the MCA and CD4+ T cells at the LE and stroma significantly associated with decreased overall survival.

A Therapeutically Actionable Protumoral Axis of Cytokines Involving IL-8, TNFα, and IL-1ß.

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1ß upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1ß induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1ß inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated. SIGNIFICANCE: IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1ß are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007.

Immune Mechanisms of Resistance to Cediranib in Ovarian Cancer.

This article investigates mechanisms of resistance to the VEGF receptor inhibitor cediranib in high-grade serous ovarian cancer (HGSOC), and defines rational combination therapies. We used three different syngeneic orthotopic mouse HGSOC models that replicated the human tumor microenvironment (TME). After 4 to 5 weeks treatment of established tumors, cediranib had antitumor activity with increased tumor T-cell infiltrates and alterations in myeloid cells. However, continued cediranib treatment did not change overall survival or the immune microenvironment in two of the three models. Moreover, treated mice developed additional peritoneal metastases not seen in controls. Cediranib-resistant tumors had intrinsically high levels of IL6 and JAK/STAT signaling and treatment increased endothelial STAT3 activation. Combination of cediranib with a murine anti-IL6 antibody was superior to monotherapy, increasing mouse survival, reducing blood vessel density, and pSTAT3, with increased T-cell infiltrates in both models. In a third HGSOC model, that had lower inherent IL6 JAK/STAT3 signaling in the TME but high programmed cell death protein 1 (PD-1) signaling, long-term cediranib treatment significantly increased overall survival. When the mice eventually relapsed, pSTAT3 was still reduced in the tumors but there were high levels of immune cell PD-1 and Programmed death-ligand 1. Combining cediranib with an anti-PD-1 antibody was superior to monotherapy in this model, increasing T cells and decreasing blood vessel densities. Bioinformatics analysis of two human HGSOC transcriptional datasets revealed distinct clusters of tumors with IL6 and PD-1 pathway expression patterns that replicated the mouse tumors. Combination of anti-IL6 or anti-PD-1 in these patients may increase activity of VEGFR inhibitors and prolong disease-free survival.

Building invitro 3D human multicellular models of high-grade serous ovarian cancer.

Three-dimensional (3D), multicellular invitro models provide a useful platform for studying human cancer biology, particularly through deconvolution of the tumor microenvironment, or where animal models do not recapitulate the human condition. Here, we detail a protocol for building human multicellular models made of patient-derived primary cells and malignant cell lines, which recapitulate features of the tumor microenvironment. This protocol is optimized for building 3D models of high-grade serous ovarian cancer omental metastasis but can be adapted for modeling other cancers. For complete details on the use and execution of this profile, please refer to Delaine-Smith et al. (2021) and Malacrida et al. (2021).

Harnessing cytokines and chemokines for cancer therapy.

During the past 40 years, cytokines and cytokine receptors have been extensively investigated as either cancer targets or cancer treatments. A strong preclinical rationale supports therapeutic strategies to enhance the growth inhibitory and immunostimulatory effects of interferons and interleukins, including IL-2, IL-7, IL-12 and IL-15, or to inhibit the inflammatory and tumour-promoting actions of cytokines such as TNF, IL-1ß and IL-6. This rationale is underscored by the discovery of altered and dysregulated cytokine expression in all human cancers. These findings prompted clinical trials of several cytokines or cytokine antagonists, revealing relevant biological activity but limited therapeutic efficacy. However, most trials involved patients with advanced-stage disease, which might not be the optimal setting for cytokine-based therapy. The advent of more effective immunotherapies and an increased understanding of the tumour microenvironment have presented new approaches to harnessing cytokine networks in the treatment of cancer, which include using cytokine-based therapies to enhance the activity or alleviate the immune-related toxicities of other treatments as well as to target early stage cancers. Many challenges remain, especially concerning delivery methods, context dependencies, and the pleiotropic, redundant and often conflicting actions of many cytokines. Herein, we discuss the lessons learnt from the initial trials of single-agent cytokine-based therapies and subsequent efforts to better exploit such agents for the treatment of solid tumours.

Stromal Cells Promote Matrix Deposition, Remodelling and an Immunosuppressive Tumour Microenvironment in a 3D Model of Colon Cancer.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. CRC develops in a complex tumour microenvironment (TME) with both mesenchymal stromal cells (MSCs) and immune infiltrate, shown to alter disease progression and treatment response. We hypothesised that an accessible, affordable model of CRC that combines multiple cell types will improve research translation to the clinic and enable the identification of novel therapeutic targets. A viable gelatine-methacrloyl-based hydrogel culture system that incorporates CRC cells with MSCs and a monocyte cell line was developed. Gels were analysed on day 10 by PCR, cytokine array, microscopy and flow cytometry. The addition of stromal cells increased transcription of matrix remodelling proteins FN1 and MMP9, induced release of tumour-promoting immune molecules MIF, Serpin E1, CXCL1, IL-8 and CXCL12 and altered cancer cell expression of immunotherapeutic targets EGFR, CD47 and PD-L1. Treatment with PD153035, an EGFR inhibitor, revealed altered CRC expression of PD-L1 but only in gels lacking MSCs. We established a viable 3D model of CRC that combined cancer cells, MSCs and monocytic cells that can be used to research the role the stroma plays in the TME, identify novel therapeutic targets and improve the transitional efficacy of therapies.

TGFBI Production by Macrophages Contributes to an Immunosuppressive Microenvironment in Ovarian Cancer.

The tumor microenvironment evolves during malignant progression, with major changes in nonmalignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein TGF beta induced (TGFBI, also known as ßig-h3). STIC lesions in the fimbria expressed high levels of TGFBI, which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFß and IL4 induced secretion of TGFBI, whereas IFNγ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvß3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro. Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated ß3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumor-promoting actions of TGFß and a potential therapeutic target. SIGNIFICANCE: Analysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer.

Pancreatic Cancer Chemotherapy Is Potentiated by Induction of Tertiary Lymphoid Structures in Mice.

BACKGROUND AND AIMS: The presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer. METHODS: Multicolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (KrasG12D, p53R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth. RESULTS: Mature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation. CONCLUSIONS: This study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology.

Modelling TGFßR and Hh pathway regulation of prognostic matrisome molecules in ovarian cancer.

In a multi-level "deconstruction" of omental metastases, we previously identified a prognostic matrisome gene expression signature in high-grade serous ovarian cancer (HGSOC) and twelve other malignancies. Here, our aim was to understand how six of these extracellular matrix (ECM) molecules, COL11A1, cartilage oligomeric matrix protein, FN1, versican, cathepsin B, and COL1A1, are upregulated in cancer. Using biopsies, we identified significant associations between TGFßR activity, Hedgehog (Hh) signaling, and these ECM molecules and studied the associations in mono-, co-, and tri-culture. Activated omental fibroblasts (OFs) produced more matrix than malignant cells, directed by TGFßR and Hh signaling cross talk. We "reconstructed" omental metastases in tri-cultures of HGSOC cells, OFs, and adipocytes. This combination was sufficient to generate all six ECM proteins and the matrisome expression signature. TGFßR and Hh inhibitor combinations attenuated fibroblast activation and gel and ECM remodeling in these models. The tri-culture model reproduces key features of omental metastases and allows study of diseased-associated ECM.

A human multi-cellular model shows how platelets drive production of diseased extracellular matrix and tissue invasion.

Guided by a multi-level "deconstruction" of omental metastases, we developed a tetra (four cell)-culture model of primary human mesothelial cells, fibroblasts, adipocytes, and high-grade serous ovarian cancer (HGSOC) cell lines. This multi-cellular model replicated key elements of human metastases and allowed malignant cell invasion into the artificial omental structure. Prompted by findings in patient biopsies, we used the model to investigate the role of platelets in malignant cell invasion and extracellular matrix, ECM, production. RNA (sequencing and quantitative polymerase-chain reaction), protein (proteomics and immunohistochemistry) and image analysis revealed that platelets stimulated malignant cell invasion and production of ECM molecules associated with poor prognosis. Moreover, we found that platelet activation of mesothelial cells was critical in stimulating malignant cell invasion. Whilst platelets likely activate both malignant cells and mesothelial cells, the tetra-culture model allowed us to dissect the role of both cell types and model the early stages of HGSOC metastases.

Chemotherapy Induces Tumor-Associated Macrophages that Aid Adaptive Immune Responses in Ovarian Cancer.

Neoadjuvant chemotherapy (NACT) may stimulate anticancer adaptive immune responses in high-grade serous ovarian cancer (HGSOC), but little is known about effects on innate immunity. Using omental biopsies from HGSOC, and omental tumors from orthotopic mouse HGSOC models that replicate the human tumor microenvironment, we studied the impact of platinum-based NACT on tumor-associated macrophages (TAM). We found that chemotherapy reduces markers associated with alternative macrophage activation while increasing expression of proinflammatory pathways, with evidence of inflammasome activation. Further evidence of a shift in TAM functions came from macrophage depletion via CSF1R inhibitors (CSF1Ri) in the mouse models. Although macrophage depletion in established disease had no impact on tumor weight or survival, CSF1Ri treatment after chemotherapy significantly decreased disease-free and overall survival. This decrease in survival was accompanied by significant inhibition of adaptive immune response pathways in the tumors. We conclude that chemotherapy skews the TAM population in HSGOC toward an antitumor phenotype that may aid adaptive immune responses, and therapies that enhance or sustain this during remission may delay relapse.

Specific Mechanisms of Chromosomal Instability Indicate Therapeutic Sensitivities in High-Grade Serous Ovarian Carcinoma.

Chromosomal instability (CIN) comprises continual gain and loss of chromosomes or parts of chromosomes and occurs in the majority of cancers, often conferring poor prognosis. Because of a scarcity of functional studies and poor understanding of how genetic or gene expression landscapes connect to specific CIN mechanisms, causes of CIN in most cancer types remain unknown. High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, is the major cause of death due to gynecologic malignancy in the Western world, with chemotherapy resistance developing in almost all patients. HGSC exhibits high rates of chromosomal aberrations and knowledge of causative mechanisms would represent an important step toward combating this disease. Here we perform the first in-depth functional characterization of mechanisms driving CIN in HGSC in seven cell lines that accurately recapitulate HGSC genetics. Multiple mechanisms coexisted to drive CIN in HGSC, including elevated microtubule dynamics and DNA replication stress that can be partially rescued to reduce CIN by low doses of paclitaxel and nucleoside supplementation, respectively. Distinct CIN mechanisms indicated relationships with HGSC-relevant therapy including PARP inhibition and microtubule-targeting agents. Comprehensive genomic and transcriptomic profiling revealed deregulation of various genes involved in genome stability but were not directly predictive of specific CIN mechanisms, underscoring the importance of functional characterization to identify causes of CIN. Overall, we show that HGSC CIN is complex and suggest that specific CIN mechanisms could be used as functional biomarkers to indicate appropriate therapy. SIGNIFICANCE: These findings characterize multiple deregulated mechanisms of genome stability that lead to CIN in ovarian cancer and demonstrate the benefit of integrating analysis of said mechanisms into predictions of therapy response.

Airway dendritic cell maturation in children exposed to air pollution.

Urban particulate matter (PM) enhances airway dendritic cell (DC) maturation in vitro. However, to date, there are no data on the association between exposure to urban PM and DC maturation in vivo. We sought to determine whether exposure of school-age children (8 to 14 y) to PM was associated with expression of CD86, a marker of maturation of airway conventional DCs (cDC). Healthy London school children underwent spirometry and sputum induction. Flow cytometry was used to identify CD86 and CCR7 expression on cDC subsets (CD1c+ cDC2 and CD141+ cDC1). Tertiles of mean annual exposure to PM ≤ 10 microns (PM10) at the school address were determined using the London Air Quality Toolkit model. Tertiles of exposure from the 409 children from 19 schools recruited were; lower (23.1 to 25.6 µg/m3, n = 138), middle (25.6 to 26.8 µg/m3, n = 126), and upper (26.8 to 31.0 µg/m3, n = 145). DC expression was assessed in 164/370 (44%) children who completed sputum induction. The proportion (%) of cDC expressing CD86 in the lower exposure tertile (n = 47) was lower compared with the upper exposure tertile (n = 49); (52% (44 to 70%) vs 66% (51 to 82%), p<0.05). There was a higher percentage of cDC1 cells in the lower tertile of exposure (6.63% (2.48 to 11.64) vs. 2.63% (0.72 to 7.18), p<0.05). Additionally; children in the lower exposure tertile had increased FEV1 compared with children in the upper tertile; (median z-score 0.15 (-0.59 to 0.75) vs. -0.21 (-0.86 to 0.48), p<0.05. Our data reveal that children attending schools in the highest areas of PM exposure in London exhibit increased numbers of "mature" airway cDCs, as evidenced by their expression of the surface marker CD86. This data is supportive of previous in vitro data demonstrating an alteration in the maturation of airway cDCs in response to exposure to pollutants.

Combining measures of immune infiltration shows additive effect on survival prediction in high-grade serous ovarian carcinoma.

BACKGROUND: In colorectal and breast cancer, the density and localisation of immune infiltrates provides strong prognostic information. We asked whether similar automated quantitation and combined analysis of immune infiltrates could refine prognostic information in high-grade serous ovarian carcinoma (HGSOC) and tested associations between patterns of immune response and genomic driver alterations. METHODS: Epithelium and stroma were semi-automatically segmented and the infiltration of CD45RO+, CD8+ and CD68+ cells was automatically quantified from images of 332 HGSOC patient tissue microarray cores. RESULTS: Epithelial CD8 [p = 0.027, hazard ratio (HR) = 0.83], stromal CD68 (p = 3 × 10-4, HR = 0.44) and stromal CD45RO (p = 7 × 10-4, HR = 0.76) were positively associated with survival and remained so when averaged across the tumour and stromal compartments. Using principal component analysis, we identified optimised multiparameter survival models combining information from all immune markers (p = 0.016, HR = 0.88). There was no significant association between PTEN expression, type of TP53 mutation or presence of BRCA1/BRCA2 mutations and immune infiltrate densities or principal components. CONCLUSIONS: Combining measures of immune infiltration provided improved survival modelling and evidence for the multiple effects of different immune factors on survival. The presence of stromal CD68+ and CD45RO+ populations was associated with survival, underscoring the benefits evaluating stromal immune populations may bring for prognostic immunoscores in HGSOC.

Cancer associated fibroblast FAK regulates malignant cell metabolism.

Emerging evidence suggests that cancer cell metabolism can be regulated by cancer-associated fibroblasts (CAFs), but the mechanisms are poorly defined. Here we show that CAFs regulate malignant cell metabolism through pathways under the control of FAK. In breast and pancreatic cancer patients we find that low FAK expression, specifically in the stromal compartment, predicts reduced overall survival. In mice, depletion of FAK in a subpopulation of CAFs regulates paracrine signals that increase malignant cell glycolysis and tumour growth. Proteomic and phosphoproteomic analysis in our mouse model identifies metabolic alterations which are reflected at the transcriptomic level in patients with low stromal FAK. Mechanistically we demonstrate that FAK-depletion in CAFs increases chemokine production, which via CCR1/CCR2 on cancer cells, activate protein kinase A, leading to enhanced malignant cell glycolysis. Our data uncover mechanisms whereby stromal fibroblasts regulate cancer cell metabolism independent of genetic mutations in cancer cells.

Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment.

Although there are many prospective targets in the tumor microenvironment (TME) of high-grade serous ovarian cancer (HGSOC), pre-clinical testing is challenging, especially as there is limited information on the murine TME. Here, we characterize the TME of six orthotopic, transplantable syngeneic murine HGSOC lines established from genetic models and compare these to patient biopsies. We identify significant correlations between the transcriptome, host cell infiltrates, matrisome, vasculature, and tissue modulus of mouse and human TMEs, with several stromal and malignant targets in common. However, each model shows distinct differences and potential vulnerabilities that enabled us to test predictions about response to chemotherapy and an anti-IL-6 antibody. Using machine learning, the transcriptional profiles of the mouse tumors that differed in chemotherapy response are able to classify chemotherapy-sensitive and -refractory patient tumors. These models provide useful pre-clinical tools and may help identify subgroups of HGSOC patients who are most likely to respond to specific therapies.