Immune response precision customized to tumor adaptive kinetics.

Despite recent notable progress in medical sciences, cancer remains a leading cause of human death worldwide, accounting for about one-quarter of human deaths in wealthy countries and about one-eighth worldwide (Ferlay et al. 2010). In fact, the progress in cancer research has been slower compared with that achieved related to other pathologies, such as cardiovascular disorders. This is mainly because of the enormous complexity of this disease, which exhibits sophisticated cellular mechanisms that are the targets of evolutionary processes driven by random genetic and epigenetic mutations.

Background: HER2-positive breast cancer is characterized by overexpression of HER2 protein and/or amplification of the HER2 gene, assessed by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. According to the 2013 ASCO/CAP guidelines on HER2 testing in breast cancer, both protein and gene status can be classified into three categories: positive, equivocal and negative. Gene-protein assay (GPA) is a newly developed technique, in which IHC and dual color ISH (DISH) are simultaneously performed on a single slide. HER2 GPA slides allow bright-field analyses of both protein and gene expression status of each cell. Breast cancer cells can be classified into nine types based on the combination of protein and gene expression with GPA. Table 1. Three by three classification of cancer cells by the HER2 GPA analysis HER2 protein 3+ (positive)2+ (equivocal)0, 1+ (negative)HER2 gene copy number≥6 (positive)ABC 4, 5 (equivocal)DEF <4 (negative)GHI Purpose: We aimed to elucidate the relationship between the results of HER2 GPA analysis and the therapeutic effects of trastuzumab-based treatment. Methods: Fifty three (53) patients with HER2-positive breast cancer, who underwent neoadjuvant trastuzumab with chemotherapy, were analyzed for HER2 status and clinical outcome. First, HER2 protein and gene status in a pre-therapeutic biopsy material from each patient were separately assessed according to the ASCO/CAP guidelines using HER2 GPA slides. Second, all cancer cells in five representative microscopic images of GPA slides were assessed to determine protein expression and to count the gene copy number at individual cell levels. Finally, we investigated the relationship between the mixture composition of the nine cell types and pathological complete response (pCR) of trastuzumab-treated breast cancer. Results: The GPA results were concordant with the results of IHC in 98% of samples, and with DISH results in 100%. Two hundred eighty nine (289) cancer cells per patient were assessed on average, ranging from 137 to 490 cells. The mean proportion of type A cancer cells was 64%, ranging from 0% to 98%. Patients who had 50% or more of type A cancer cells achieved pCR in 72% of cases (28/39), while patients with less than 50% of type A cancer cells achieved pCR in 7% of cases (1/14) (P<0.0001). The mean proportion of type C cancer cells was 7%, ranging from 0% to 60%. Patients who had 10% or more of type C cancer cells achieved pCR in 10% of cases (1/10), while patients with less than 10% of type C cancer cells achieved pCR in 65% of cases (28/43) (P=0.0016). Conclusion: HER2 GPA analyses can more precisely predict therapeutic effect of trastuzumab-based treatment in primary HER2-positive breast cancer. Our study suggests that the HER2 GPA approach would further improve precision therapy in HER2-positive breast cancer. Citation Format: Horii R, Nitta H, Ito Y, Iwase T, Ohno S, Akiyama F. Simultaneous analyses of HER2 gene and protein status can more precisely predict pathological complete response (pCR) to neoadjuvant trastuzumab with chemotherapy in primary HER2-positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-08.

BackgroundWe test whether the phenotypic variance of symbionts (Symbiodinium) in corals is closely related with the capacity of corals to acclimatize to increasing seawater temperatures. Moreover, we assess whether more specialist symbionts will increase within coral hosts under ocean warming. The present study is only applicable to those corals that naturally have the capacity to support more than one type of Symbiodinium within the lifetime of a colony; for example, Montastraea annularis and Montastraea faveolata. Methodology/Principal FindingsThe population dynamics of competing Symbiodinium symbiont populations were projected through time in coral hosts using a novel, discrete time optimal–resource model. Models were run for two Atlantic Ocean localities. Four symbiont populations, with different environmental optima and phenotypic variances, were modeled to grow, divide, and compete in the corals under seasonal fluctuations in solar insolation and seawater temperature. Elevated seawater temperatures were input into the model 1.5°C above the seasonal summer average, and the symbiont population response was observed for each location. The models showed dynamic fluctuations in Symbiodinium populations densities within corals. Population density predictions for Lee Stocking Island, the Bahamas, where temperatures were relatively homogenous throughout the year, showed a dominance of both type 2, with high phenotypic variance, and type 1, a high-temperature and high-insolation specialist. Whereas the densities of Symbiodinium types 3 and 4, a high-temperature, low-insolation specialist, and a high-temperature, low-insolation generalist, remained consistently low. Predictions for Key Largo, Florida, where environmental conditions were more seasonally variable, showed the coexistence of generalists (types 2 and 4) and low densities of specialists (types 1 and 3). When elevated temperatures were input into the model, population densities in corals at Lee Stocking Island showed an emergence of high-temperature specialists. However, even under high temperatures, corals in the Florida Keys were dominated by generalists.Conclusions/SignificancePredictions at higher seawater temperatures showed endogenous shuffling and an emergence of the high-temperature Symbiodinium specialists, even though their phenotypic variance was low. The model shows that sustaining these “hidden” specialists becomes advantageous under thermal stress conditions, and shuffling symbionts may increase the corals' capacity to acclimatize but not adapt to climatechange–induced ocean warming.

Emerging evidence suggests that the SAM pointed domain containing ETS transcription factor (SPDEF) plays a significant role in tumorigenesis in prostate, breast, colon, and ovarian cancer. However, there are no in vivo studies with respect to the role of SPDEF in tumor metastasis. The present study examined the effects of SPDEF on tumor cell metastasis using prostate tumor cells as a model. Utilizing two experimental metastasis models, we demonstrate that SPDEF inhibits cell migration and invasion in vitro and acts a tumor metastasis suppressor in vivo. Using stable expression of SPDEF in PC3-Luc cells and shRNA-mediated knockdown of SPDEF in LNCaP-Luc cells, we demonstrate for the first time that SPDEF diminished the ability of disseminated tumors cells to survive at secondary sites and establish micrometastases. These effects on tumor metastasis were not a result of the effect of SPDEF on cell growth as SPDEF expression had no effect on cell growth in vitro or subcutaneous tumor xenograft-growth in vivo. Transcriptional analysis of several genes associated with tumor metastasis, invasion, and the epithelial-mesenchymal transition demonstrated that SPDEF expression selectively down-regulated MMP9 and MMP13 in prostate cancer cells. Further analysis indicated that forced MMP9 or MMP13 expression rescued the invasive phenotype in SPDEF expressing PC3 cells in vitro, suggesting that the effects of SPDEF on tumor invasion are mediated, in part, through the suppression of MMP9 and MMP13 expression. These results demonstrate for the first time, in any system, that SPDEF functions as a tumor metastasis suppressor in vivo.

This review describes the use of ultrasound for inducing and retaining cell-cell contact in multi-well microplates combined with live-cell fluorescence microscopy. This platform has been used for studying the interaction between natural killer (NK) cells and cancer cells at the level of individual cells. The review includes basic principles of ultrasonic particle manipulation, design criteria when building a multi-well microplate device for this purpose, biocompatibility aspects, and finally, two examples of biological applications: Dynamic imaging of the inhibitory immune synapse, and studies of the heterogeneity in killing dynamics of NK cells interacting with cancer cells.

<div>Abstract<p>Subpopulations of cancer stem–like cells (CSC) are thought to drive tumor progression and posttreatment recurrence in multiple solid tumors. However, the mechanisms that maintain stable proportions of self-renewing CSC within heterogeneous tumors under homeostatic conditions remain poorly understood. Progastrin is a secreted peptide that exhibits tumor-forming potential in colorectal cancer, where it regulates pathways known to modulate colon CSC behaviors. In this study, we investigated the role of progastrin in regulating CSC phenotype in advanced colorectal cancer. Progastrin expression and secretion were highly enriched in colon CSC isolated from human colorectal cancer cell lines and colon tumor biopsies. Progastrin expression promoted CSC self-renewal and survival, whereas its depletion by RNA interference–mediated or antibody-mediated strategies altered the homeostatic proportions of CSC cells within heterogeneous colorectal cancer tumors. Progastrin downregulation also decreased the frequency of ALDH<sup>high</sup> cells, impairing their tumor-initiating potential, and inhibited the high glycolytic activity of ALDH<sup>high</sup> CSC to limit their self-renewal capability. Taken together, our results show how colorectal CSC maintain their tumor-initiating and self-renewal capabilities by secreting progastrin, thereby contributing to the tumor microenvironment to support malignancy. <i>Cancer Res; 76(12); 3618–28. ©2016 AACR</i>.</p></div>

<div>Abstract<p>Subpopulations of cancer stem–like cells (CSC) are thought to drive tumor progression and posttreatment recurrence in multiple solid tumors. However, the mechanisms that maintain stable proportions of self-renewing CSC within heterogeneous tumors under homeostatic conditions remain poorly understood. Progastrin is a secreted peptide that exhibits tumor-forming potential in colorectal cancer, where it regulates pathways known to modulate colon CSC behaviors. In this study, we investigated the role of progastrin in regulating CSC phenotype in advanced colorectal cancer. Progastrin expression and secretion were highly enriched in colon CSC isolated from human colorectal cancer cell lines and colon tumor biopsies. Progastrin expression promoted CSC self-renewal and survival, whereas its depletion by RNA interference–mediated or antibody-mediated strategies altered the homeostatic proportions of CSC cells within heterogeneous colorectal cancer tumors. Progastrin downregulation also decreased the frequency of ALDH<sup>high</sup> cells, impairing their tumor-initiating potential, and inhibited the high glycolytic activity of ALDH<sup>high</sup> CSC to limit their self-renewal capability. Taken together, our results show how colorectal CSC maintain their tumor-initiating and self-renewal capabilities by secreting progastrin, thereby contributing to the tumor microenvironment to support malignancy. <i>Cancer Res; 76(12); 3618–28. ©2016 AACR</i>.</p></div>

Sorghum downy mildew (SDM), caused by the biotrophic fungi Peronosclerospora sorghi, threatens maize production worldwide, including India. To identify quantitative trait loci (QTL) associated with resistance to SDM, we used a recombinant inbred line (RIL) population derived from a cross between resistant inbred line UMI936 (w) and susceptible inbred line UMI79. The RIL population was phenotyped for SDM resistance in three environments [E1-field (Coimbatore), E2-greenhouse (Coimbatore), and E3-field (Mandya)] and also utilized to construct the genetic linkage map by genotyping by sequencing (GBS) approach. The map comprises 1516 SNP markers in 10 linkage groups (LGs) with a total length of 6924.7 cM and an average marker distance of 4.57 cM. The QTL analysis with the phenotype and marker data detected nine QTL on chromosome 1, 2, 3, 5, 6, and 7 across three environments. Of these, QTL namely qDMR1.2, qDMR3.1, qDMR5.1, and qDMR6.1 were notable due to their high phenotypic variance. qDMR3.1 from chromosome 3 was detected in more than one environment (E1 and E2), explaining the 10.3% and 13.1% phenotypic variance. Three QTL, qDMR1.2, qDMR5.1, and qDMR6.1 from chromosomes 1, 5, and 6 were identified in either E1 or E3, explaining 15.2%–18% phenotypic variance. Moreover, genome mining on three QTL (qDMR3.1, qDMR5.1, and qDMR6.1) reveals the putative candidate genes related to SDM resistance. The information generated in this study will be helpful for map-based cloning and marker-assisted selection in maize breeding programs.

BackgroundAcquired resistance is a major limiting factor for durable T cell therapies in solid tumors. Antigen escape pathways such as insufficient antigen coverage or loss of target antigen remain major...

Long non-coding RNAs are sequences longer than 200 nucleotides that are involved in different normal and abnormal biological processes exerting their effect on proliferation and differentiation, among other cell features. Functionally, lncRNAs can regulate gene expression within the cells by acting at transcriptional, post-transcriptional, translational, or post-translational levels. However, in pathological conditions such as cancer, the expression of these molecules is deregulated, becoming elements that can help in the acquisition of tumoral characteristics in the cells that trigger carcinogenesis and cancer progression. Specifically, in gallbladder cancer (GBC), recent publications have shown that lncRNAs participate in the acquisition of an aggressive phenotype in cancer cells, allowing them to acquire increased malignant capacities such as chemotherapy resistance or metastasis, inducing a worse survival in these patients. Furthermore, lncRNAs are useful as prognostic and diagnostic biomarkers since they have been shown to be differentially expressed in tumor tissues and serum of individuals with GBC. Therefore, this review will address different lncRNAs that could be promoting malignant phenotypic characteristics in GBC cells and lncRNAs that may be useful as markers due to their capability to predict a poor prognosis in GBC patients.

Objective: Since scRNA-seq is an effective tool to study tumor heterogeneity, this paper intends to reveal the differences of cervical cancer in patients at the individual cell level by scRNA-seq, and focus on the biological functions of cancer-associated fibroblasts (CAFs) in cervical cancer, facilitating the provision of a new interpretation of the heterogeneity of the microenvironment of cervical cancer, and an in-depth exploration of the pathogenesis of cervical cancer as well as pursuit of effective means of treatment intake.Methods: 3 cervical cancer specimens were collected by clinical surgery for single-cell RNA sequencing. Cell suspensions of fresh cervical cancer tissues were prepared, and cDNA libraries were created and sequenced on the machine. Furthermore, the sequencing data were analyzed using bioinformatics, including descending clustering of cells, identification of cell populations, mimetic time series analysis, inferCNV, cell communication analysis, and identification of transcription factors.Results: A total of 9 cell types were identified, encompassing T cells, epithelial cells, smooth muscle cells, CAFs, endothelial cells, macrophages, B cells, lymphocytes, and plasma cells. CAFs were further divided into three cell subtypes, named type1 cells, type2 cells, and type3 cells. With key transcription factors for the three cells, TCF21, ZC3H11A, and MYEF2 obtained, this research revealed the communication relationship between CAFs and several other cells, and found an important role of CAFs in the MK signaling pathway.Conclusions: scRNA-seq technology contributed to exploring the tumor heterogeneity of cervical cancer more deeply, and also further gaining insight into the biological functions of CAFs in cervical cancer.

Chromosomal instability (CIN), characterized by pervasive copy number alterations (CNAs), significantly contributes to cancer progression and therapeutic resistance. CNAs drive intratumoral genetic heterogeneity, creating distinct subpopulations whose interactions shape tumor evolution through frequency-dependent selection. Here, we introduce, ECO-K (Ecological-Karyotypes), an inverse game theory framework that infers subpopulation interactions from longitudinal single-cell whole genome sequencing data. Applying this approach to serially-passaged, triple-negative breast cancer (TNBC) cell lines and patient-derived xenografts (PDXs), we systematically identified frequency-dependent selection dynamics governed by karyotypic diversity. Notably, in one PDX lineage, we found consistent evidence of karyotypically defined subpopulations acting as interaction hubs, associated specifically with chromosome 1 loss and chromosome 14p gain. Our framework provides testable predictions of intratumoral ecological dynamics, highlighting opportunities to strategically target key subpopulations to disrupt tumor evolution.

In Breast Cancer, Tumor-infiltrating lymphocyte (TIL) motility has been reported that it is reflecting tumor microenvironment response by adoptive cell therapy (ACT). ACT is well-known to be best applied for suppressing cancer cell growth with immune response and it also was well-known the immunotherapy. However, ACT response tracking by general analysis method included immunohistochemistry and flow cytometry was provided limited information for cancer therapy in recent years. Therefore, by assessing relationship immunotherapy marker, PD-L1, known as immune check point inhibitor and TIL using intravital microscopy in breast cancer model, we also imaged whether TIL response was related to immunotherapy following dependent on time point in a various breast cancer cell implanted mouse model such as 4T1 (mouse breast cancer cell), MDA-MB-231 (human triple negative breast cancer cell) and MCF7 (human breast cancer cell). These images result in PD-L1 expression was decreased in cancer cell implanted until 10days, whereas TIL expression increased in a various breast cancer cell when it is measuring displacement length, track mean velocity and confinement ratio called tumor microenvironment. These results indicated that we can imaged immune response related to TIL response in breast cancer cell line by intravital microscopy instead of general microscopy. It means that intravital microscope was an advanced approach technique to understand cancer mechanism accompanied by the immune response and established the method of high-resolution intravital cellular visualization compared to a general histopathological method for evaluating immune response in cancer tissue Citation Format: Hyunseok Kim, Soyeon Ahn, Hyungjin Kwon, Hsuan-Ju Chen, Inwon Park, Pilhan Kim, Akyildiz Kubra. An advanced approach technique for tracking point between immune check point inhibitor and cancer cell mechanism in immune response motility by infiltration a breast cancer cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2397.

Background: A primary limitation of PD-1 inhibitors in non-small cell lung cancer (NSCLC) arises from their inability to act on 'cold' tumors without tumor-reactive T cells, necessitating alternative approaches. Adoptive cell therapy (ACT) using (CAR)-engineered cells, strives to enhance antitumor immunity but faces several challenges such as identifying safe antigens, tumor heterogeneity, antigen escape, cell trafficking, and T cell persistence. To address these issues, we explored a new source of T cells from the benign tumor draining lymph nodes (tdLNs) of NSCLC patients. Our initial results have shown that tdLNs is a reservoir for of tumor-relevant 'stem-like' T cells. We posit that employing these pluripotent T cells for ACT could achieve significant tumor rejection in NSCLC. Methods: Resected tumors, tdLN, non-draining (ndLN), and blood from NSCLC patients, as well as a syngeneic murine lung cancer model (344SQ), underwent analysis. T cells were profiled using flow cytometry, cytotoxicity and proliferation assays. TCR and single cell (sc)RNA sequencing assessed clonal expansion, diversity, and transcriptional profiles of tumor-relevant T cells. T cells were then transduced with an ICAM-1 targeting CAR, in vivo efficacy was evaluated in an A549 murine lung cancer model. Results: T cell subsets with stem-cell memory characteristics, as indicated by PD-1+, TCF1hi, CXCR5+, and CD8+ expression, which were not significantly found in the tumor or PB. These T cells exhibited progenitor-like transcriptional signatures, enhanced SELL and TCF-1 expression, fewer exhaustion markers, and superior in vitro proliferation compared to TILs from both a murine lung cancer model and patient-derived tissues. scRNA sequencing, coupled with TCR "tumor matching" (TM) techniques, exposed a rich clonal diversity of tumor-relevant clones within tdLNs, which showcased a broader transcriptional memory profile and distinct CD4+ and CD8+ phenotypes. Upon analyzing the top 100 expanded (n>3) TM clones, 47 featured the presence of tdLN-derived T cells, covering progenitor, stem cell-like, and central memory clusters. T cell subsets were then transduced with a CAR targeting ICAM-1—a cell surface protein frequently overexpressed in NSCLC tumors. Manufacturing protocol yielded high transduction efficiency and T cell expansion within two weeks in 6/6 patients, consistent with PB-derived CAR T cells and on par with the optimal dosing requirements of an ICAM-1 CAR Phase I trial (NCT04420754). tdLN-CAR T cells showed potent antitumor efficacy compared to the control in an aggressive NSCLC murine model (Median survival 103d vs 66d; respectively; p=0.006). Conclusions: This represents the first reported use of T cells from tdLN for genetically engineered ACT. The data indicate that modifying antigen-experienced, stem-like T cells from tdLN with CAR is a promising and efficient method in a NSCLC murine model, warranting further research. Citation Format: Tatiana Delgado Cruz, Yanping Yang, Rachel Honigsberg, Geoffrey Markowitz, Nasser K Altorki, Vivek Mittal, Moonsoo M. Jin, Jonathan Villena-Vargas. T cells selected from lymph node acquisition for adoptive cell therapyin NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2.

Breast cancer (BC) is a heterogeneous disease with evolving genetic alterations and expressions of receptor proteins. Intratumoral heterogeneity (ITH) is considered to be a resistance factor in response to targeted therapies. The current single-slide, single-marker immunohistochemistry techniques cannot accurately assess ITH at the individual cancer cell level. In this study, we develop a novel brightfield multiplex assay to simultaneously assess estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) protein markers, together with the HER2 gene and the centromere of chromosome 17 (CEP17) copy numbers, using a single tissue section. The data presented herein demonstrate heterogeneous cancer cell subpopulations in 11 HER2-positive/ER-positive (HER2+/ER+) tumors among 33 BCs analyzed immunohistochemically (HER2 score of 2+ or 3+). The predominant cancer cell subpopulation was HER2+/ER- (50.18%), followed by HER2+/ER+ (39.05%), HER2-/ER+ (4.26%), ER- with HER2 microheterogeneity cancer cells (3.58%), and ER+ with HER2 microheterogeneity cancer cells (2.93%). The three other tumor subtypes, namely, HER2-/ER+, HER2+/ER-, and HER2-/ER-, were more homogeneous, representing 82.59%, 99.22%, and 100% of cancer cells, respectively. This novel assay revealed that HER2+ cancer cells were more predominant than ER+ cancer cells in HER2+/ER+ tumors and provided new insights toward our understanding of BC carcinogenesis.