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Aberrant expression of viral-like repeat elements is a common feature of epithelial cancers, and the substantial diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering independent of tissue origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that human satellite II (HSATII) satellite repeat expression was highly associated with epithelial-mesenchymal transition (EMT) and anticorrelated with IFN-response genes indicative of a more aggressive phenotype. SATII expression--and its correlation with EMT and anticorrelation with IFN-response genes-- was also found in ovarian cancer RNA-Seq data and was associated with significantly shorter survival in a second independent cohort of patients with ovarian cancer. Repeat RNAs were enriched in tumor-derived extracellular vesicles capable of stimulating monocyte-derived macrophages, demonstrating a mechanism that alters the tumor microenvironment with these viral-like sequences. Targeting of HSATII with antisense locked nucleic acids stimulated IFN response and induced MHC I expression in ovarian cancer cell lines, highlighting a potential strategy of modulating the repeatome to reestablish antitumor cell immune surveillance., Introduction Repetitive elements make up approximately 50% of the human genome (1, 2), and their aberrant expression has been described across a wide range ofcancers (3-5). Repeat suppression is achieved [...]

Circulating tumor cells (CTCs) are extremely rare in the blood, yet they account for metastasis. Notably, it was reported that CTC clusters (CTCCs) can be 50–100 times more metastatic than single CTCs, making them particularly salient as a liquid biopsy target. Yet they can split apart and are even rarer, complicating their recovery. Isolation by filtration risks loss when clusters squeeze through filter pores over time, and release of captured clusters can be difficult. Deterministic lateral displacement is continuous but requires channels not much larger than clusters, leading to clogging. Spiral inertial focusing requires large blood dilution factors (or lysis). Here, we report a microfluidic chip that continuously isolates untouched CTC clusters from large volumes of minimally (or undiluted) whole blood. An array of 100 μm-wide channels first concentrates clusters in the blood, and then a similar array transfers them into a small volume of buffer. The microscope-slide-sized PDMS device isolates individually-spiked CTC clusters from >30 mL per hour of whole blood with 80% efficiency into enumeration (fluorescence imaging), and on-chip yield approaches 100% (high speed video). Median blood cell removal (in base-10 logs) is 4.2 for leukocytes, 5.5 for red blood cells, and 4.9 for platelets, leaving less than 0.01% of leukocytes alongside CTC clusters in the product. We also demonstrate that cluster configurations are preserved. Gentle, high throughput concentration and separation of circulating tumor cell clusters from large blood volumes will enable cluster-specific diagnostics and speed the generation of patient-specific CTC cluster lines. [ABSTRACT FROM AUTHOR]

The metabolic gene isocitrate dehydrogenase 1 (IDH1) is commonly mutated in lower grade glioma (LGG) and secondary glioblastoma (GBM). Regulatory T cells (Tregs) play a significant role in the suppression of antitumor immunity in human glioma. Given the importance of Tregs in the overall framework of designing immune-based therapies, a better understanding on their association with IDH mutational status remains of critical clinical importance. Using multispectral imaging analysis, we compared the incidence of Tregs in IDH-mutant and IDH wild-type glioma from patient tumor samples of LGG. An orthotopic IDH-mutant murine model was generated to evaluate the role of mutant IDH on Treg infiltration by immunohistochemistry. When compared to IDH wild-type controls, Tregs are disproportionally underrepresented in mutant disease, even when taken as a proportion of all infiltrating T cells. Our findings suggest that therapeutic agents targeting Tregs may be more appropriate in modulating the immune response to wild-type disease. [ABSTRACT FROM AUTHOR]

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies. [ABSTRACT FROM AUTHOR]

We describe a home-built apertureless near-field scanning optical microscope and present preliminary results of its operation. Raman scattering from samples of polydiacetylene para-toluene sulphonate, and two-photon-induced fluorescence from crystallites of coumarin I dye are strongly enhanced in the presence of a sharp gold-coated atomic force microscope tip. We verify the dependence of the scattered intensity on the polarization of the incident beam relative to the tip axis. Finally, we show near-field fluorescence images taken in the presence of a strong far-field background whose spatial resolution is limited by the size of the tip. © 2001 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Immune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. Here, we demonstrate that Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of emerging anti-cancer Antibody Drug Conjugates (ADCs), controls barrier-mediated immune exclusion in Triple-Negative Breast Cancer (TNBC) through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and strongly associated with outcomes in TNBC. Loss-of-function and reconstitution experiments demonstrate TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. Employing a humanized TROP2 syngeneic TNBC model, we show that TROP2 targeting via hRS7, the antibody component of Sacituzumab govitecan (SG), enhances the anti-PD1 response associated with improved T cell accessibility and effector function. Correspondingly, TROP2 expression is highly associated with lack of response to anti-PD1 therapy in human breast cancer. Thus, TROP2 controls an immune exclusion program that can be targeted to enhance immunotherapy response., Competing Interests: D.T.T. has received consulting fees from ROME Therapeutics, Sonata Therapeutics, Tekla Capital, and abrdn. D.T.T. is a founder and has equity in ROME Therapeutics, PanTher Therapeutics and TellBio, Inc., which is not related to this work. D.T.T. is on the advisory board for ImproveBio, Inc. D.T.T. has received honorariums from AstraZeneca, Moderna, and Ikena Oncology that are not related to this work. D.T.T. receives research support from ACD-Biotechne, AVA LifeScience GmbH, Incyte Pharmaceuticals, and Sanofi, which was not used in this work. D.T.T.’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. S.J.I.: Institutional support from Genentech and Astra Zeneca. L.M.S.: Consultant/advisory board: Novartis, Daiichi Pharma, Astra Zeneca, Eli Lilly, Precede, Seagen; Institutional research support: Merck, Genentech, Gilead, Eli Lilly. A.B.: Consultant/advisory board: Pfizer, Novartis, Genentech, Merck, Radius Health; Immunomedics/Gilead, Sanofi, Daiichi Pharma/Astra Zeneca, Phillips, Eli Lilly, Foundation Medicine; Contracted Research/Grant (to institution): Genentech, Novartis, Pfizer, Merck, Sanofi, Radius Health, Immunomedics/Gilead, Daiichi Pharma/Astra Zeneca, Eli Lilly. L.W.E.: Consultant to Mersana, Inc.; Consultant to Kisoji Biotech; Consultant to Astra Zeneca; Consultant to Gilead; Sponsored Research Agreement with Sanofi. The other authors declare no competing interests.

Purpose: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells)., Experimental Design: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids., Results: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors., Conclusions: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer., (©2024 American Association for Cancer Research.)

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring., Significance: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 μL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489., (©2023 The Authors; Published by the American Association for Cancer Research.)

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements., Significance: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397., (©2022 American Association for Cancer Research.)

Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases, but not lung and liver metastases, exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-MET protein. Suppression of c-MET impaired tumor growth in the bone. Intratumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities coexisting with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in patients with metastatic prostate and breast cancer. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single-cell proteomics indicate that microenvironment- and cell state-dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors., Significance: Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies., (©2022 American Association for Cancer Research.)

RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (ΔCDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization. These changes were RB dependent, were driven by specific isoforms of monophosphorylated RB, and required known RB-associated activities. ΔCDK-RB altered physical interactions between RB-bound genomic loci, but the RB-induced changes in chromosome architecture were unaffected by dominant-negative DP1. The RB-induced changes appeared to be widespread and influenced chromosome localization within nuclei. Gene expression profiles revealed that the dispersion phenotype was associated with an increased autophagy response. We infer that, after cell cycle arrest, RB acts through noncanonical mechanisms to significantly change nuclear organization, and this reorganization correlates with transitions in cellular state., (© 2022 Krishnan et al.)

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF -mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin ( TF ), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities. This article is highlighted in the In This Issue feature, p. 521 ., (©2020 American Association for Cancer Research.)

Introduction: The expression of LGR5, a known stem cell marker, is poorly understood in Barrett's esophagus (BE) and related neoplasia. The aim of this study was to evaluate LGR5 in BE and related neoplasia and to evaluate its utility as a potential biomarker of progression to advanced neoplasia., Methods: We evaluated total 137 patients, including 119 with BE and 18 with normal gastroesophageal mucosa for expression of LGR5 using RNA in situ hybridization; this also included 28 progressors and 30 nonprogressors. The LGR5 stain was evaluated using 1 qualitative and 2 quantitative parameters, using manual and automated platforms., Results: Surface LGR5 expression was mainly seen in high-grade dysplasia (12/18) compared with low-grade dysplasia (1/8) and nondysplastic BE (0/17) (P < 0.0001). In contrast to nondysplastic BE, low- and high-grade dysplasia showed a higher percentage of mean number of LGR5-positive crypts per patient (P < 0.0001) and an increase in the mean number of LGR5 transcripts per cell (P < 0.0001). The mean percentage of LGR5-positive crypts per patient and the mean number of LGR5 transcripts per cell were also significantly higher in nondysplastic BE from progressor compared with nonprogressor (P < 0.0001, P = 0.014). The sensitivity and specificity of LGR5 for distinguishing progressor from nonprogressor were 50% and 87%, respectively., Discussion: BE-related advanced neoplasia shows an expansion of the LGR5-positive cellular compartment, supporting its role as a stem cell marker in this disease. Quantitative LGR5 expression and surface epithelial reactivity are novel biomarkers of increased risk of progression to advanced neoplasia in BE., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Cryopreservation is of significance in areas including tissue engineering, regenerative medicine, and organ transplantation. We investigated endothelial cell attachment and membrane integrity in a microvasculature model at high subzero temperatures in the presence of extracellular ice. The results show that in the presence of heterogeneous extracellular ice formation induced by ice nucleating bacteria, endothelial cells showed improved attachment at temperature minimums of -6 °C. However, as temperatures decreased below -6 °C, endothelial cells required additional cryoprotectants. The glucose analog, 3- O -methyl-D-glucose (3-OMG), rescued cell attachment optimally at 100 mM (cells/lane was 34, as compared to 36 for controls), while 2% and 5% polyethylene glycol (PEG) were equally effective at -10 °C (88% and 86.4% intact membranes). Finally, endothelialized microchannels were stored for 72 h at -10 °C in a preservation solution consisting of the University of Wisconsin (UW) solution, Snomax, 3-OMG, PEG, glycerol, and trehalose, whereby cell attachment was not significantly different from unfrozen controls, although membrane integrity was compromised. These findings enrich our knowledge about the direct impact of extracellular ice on endothelial cells. Specifically, we show that, by controlling the ice nucleation temperature and uniformity, we can preserve cell attachment and membrane integrity. Further, we demonstrate the strength of leveraging endothelialized microchannels to fuel discoveries in cryopreservation of thick tissues and solid organs., Competing Interests: Notes The authors declare the following competing financial interest(s): S.N.T., M.T., S.L.S., K.U., and L.W. are inventors on several provisional patents on the topic of cryopreservation of cells, tissues, and organs, including high subzero preservation. Further, K.U. has a financial interest in Organ Solutions, a company focused on developing organ preservation technology. The interests of all researchers are managed by the MGH and Partners HealthCare in accordance with their conflict of interest policies.

We present an approach to adaptively adjust the spectral window sizes for optical spectra feature extraction. Previous studies extracted features from spectral windows of a fixed width. In our algorithm, piecewise linear regression is used to adaptively adjust the window sizes to find the maximum window size with reasonable linear fit with the spectrum. This adaptive windowing technique ensures the signal linearity in defined windows; hence, the adaptive windowing technique retains more diagnostic information while using fewer windows. This method was tested on a data set of diffuse reflectance spectra of oral mucosa lesions. Eight features were extracted from each window. We performed classifications using linear discriminant analysis with cross-validation. Using windowing techniques results in better classification performance than not using windowing. The area under the receiver-operating-characteristics curve for windowing techniques was greater than a nonwindowing technique for both normal versus mild dysplasia (MD) plus severe high-grade dysplasia or carcinama (SD) (MD+SD) and benign versus MD+SD. Although adaptive and fixed-size windowing perform similarly, adaptive windowing utilizes significantly fewer windows than fixed-size windows (number of windows per spectrum: 8 versus 16). Because adaptive windows retain most diagnostic information while reducing the number of windows needed for feature extraction, our results suggest that it isolates unique diagnostic features in optical spectra.

We report the development and evaluation of a simple compact probe that incorporates multiple beveled fibers for depth sensitive detection of spectroscopic signals in vivo. We evaluated three probes with bevel angles 35, 40, and 45 degrees for their collection efficiency and depth resolution using a thin highly scattering white substrate and found that a 40 degree bevel provides the best characteristics for depth-resolved spectroscopy. The depth sensitivity of the probe with 40 degree beveled fibers was then evaluated using multilayer phantoms with scattering properties mimicking precancerous tissue and in vivo on normal human oral mucosa. The results demonstrate that the use of multiple beveled fibers has the capability to simultaneously collect scattering spectra from a range of depths within epithelial tissue that has the potential to provide further significant improvement of detection and monitorin of epithelial precancers.

We report the results of an oral cavity pilot clinical trial to detect early precancer and cancer using a fiber optic probe with obliquely oriented collection fibers that preferentially probe local tissue morphology and heterogeneity using oblique polarized reflectance spectroscopy (OPRS). We extract epithelial cell nuclear sizes and 10 spectral features. These features are analyzed independently and in combination to assess the best metrics for separation of diagnostic classes. Without stratifying the data according to anatomical location or level of keratinization, OPRS is found to be sensitive to four diagnostic categories: normal, benign, mild dysplasia, high-grade dysplasia, and carcinoma. Using linear discriminant analysis, separation of normal from high-grade dysplasia and carcinoma yield a sensitivity and specificity of 90 and 86%, respectively. Discrimination of morphologically similar lesions such as normal from mild dysplasia is achieved with a sensitivity of 75% and specificity of 73%. Separation of visually indistinguishable benign lesions from high-grade dysplasia and carcinoma is achieved with good sensitivity (100%) and specificity (85%), while separation of benign from mild dysplasia gives a sensitivity of 92% and a specificity of 69%. These promising results suggest that OPRS has the potential to aid screening and diagnosis of oral precancer and cancer.

We compared the use of linear discriminant analysis and an artificial neural network for classifying optical spectral measurements of oral sites. Consistent with studies of optical spectroscopy for diagnosis of other forms of cancer, our results suggest that a non-linear classifier may be warranted. Our study also demonstrated that the classifiers were better able to distinguish between sites that were more histopathologically distinct.

Early detection of cancer and its curable precursors remains the best way to ensure patient survival and quality of life. Thus, highly selective, sensitive and cost-effective screening and diagnostic techniques to identify curable pre-cancerous lesions are desperately needed. Precancers are characterized by increased nuclear size, increased nuclear/cytoplasmic ratio, hyperchromasia and pleomorphism, which currently can only be assessed through an invasive, painful biopsy. Here, we describe the development of a non-invasive optical technique based on polarized reflectance spectroscopy that has the potential to provide in real time diagnostically useful information for pre-cancer detection. Our results demonstrate that polarized reflectance spectroscopy can be used to selectively detect the size-dependent scattering characteristics of nuclei in vivo. We gradually progress from cell suspensions to realistic three-dimensional tissue models of epithelium, then to cervical biopsies and, finally to in vivo studies on normal volunteers and clinical patients.