IntroductionHand-Foot Syndrome (HFS) is a common side effect in patients undergoing chemotherapy, particularly with Doxorubicin and Docetaxel and can substantially impair quality of life. This study aimed to assess the feasibility and acceptability of using Ice Plant Intensive Cream (Mesembryanthemum crystallinum) to prevent HFS in breast cancer patients, within a predefined recruitment target of 36 participants.MethodsIn this randomized controlled pilot study, patients with breast cancer scheduled to receive treatment with doxorubicin or docetaxel were enrolled. Participants were randomly assigned either to an intervention group, which followed standard HFS prevention plus Ice Plant Intensive Cream to the hands and feet three times daily during chemotherapy, or a control group that received standard HFS prevention alone. Feasibility endpoints included recruitment rate, inclusion rate, retention, adherence to the intervention, and completeness of data collection. Secondary exploratory endpoints included HFS severity, skin-related symptoms, and quality of life.ResultsBetween January and November 2023, 330 patients were screened during tumor board meetings, of whom 58 met the eligibility criteria. Ultimately, 13 patients (22.4% of eligible) consented and were enrolled, indicating that neither the predefined recruitment target nor the randomization feasibility criterion were met. Most non-participation was attributable to patient overload during the diagnostic phase or initiation of chemotherapy before contact. Once enrolled, feasibility metrics related to retention, adherence, and data completeness were achieved, with a dropout rate of 15%, protocol adherence of 100% among participants who completed the study, and more than 90% completeness of scheduled assessments.ConclusionThe overall study was not feasible as designed, primarily due to recruitment limitations. However, once patients were enrolled, intervention procedures, data collection, and protocol adherence were fully feasible. Optimization of recruitment timing and earlier patient engagement during the diagnostic phase are recommended for future confirmatory trials.Clinical Trial Registrationhttps://clinicaltrials.gov/study/NCT05755646, identifier NCT05755646.

Abstract Ovarian cancer is a complex and heterogeneous disease and the major cause of death among women with gynecological cancers. While patients usually respond well to the platinum-based first-line chemotherapy, the disease often becomes increasingly resistant to the treatment. The tumor microenvironment (TME) plays an important role in tumor development and drug resistance. Characterizing the TME of ovarian cancer after drug treatment is essential to identify molecular mechanisms that allow residual tumor cells to survive chemotherapy in ovarian cancer. We have established a preclinical model using precision-cut tumor slices (PCTS) with 250 µm thickness which preserves the TME of solid tumors with their heterogeneous cell composition during cultivation. The tumor morphology, viability and heterogeneity in terms of tumor and stromal cells as well as the immune compartment are preserved within the system. Based on the PCTS model, we also established a method to perform single-cell RNA sequencing (scRNA-seq) of the PCTS after cultivation and drug treatment to characterize the cellular features and dynamic relationships of different cell populations in the TME after drug treatment. Over 20 cell subtypes including different clusters of epithelial cells, immune cells and fibroblasts can be identified in PCTS after cisplatin treatment through the scRNA-seq analysis. This enables further deeper analysis of each cell subgroup in the TME after drug treatment. In response to cisplatin treatment, patients PCTS showed an individual induction of PD-L1 in different cell types. Additionally, multiplex immunofluorescence (mIF) stainings of the PCTS were performed to spatially trace the response of the TME to drug treatment. The mIF staining allows the simultaneous detection of up to 6 different markers on one FFPE tissue section. Images were analyzed using a machine-learning based workflow, which allows the comparison of biomarker expression and immune cell spatial distribution in the PCTS stromal and tumor areas before and after treatment. Combining the PCTS as a preclinical model with scRNA-seq and mIF staining analysis allows us to bridge the cellular characteristics and cellular spatial distribution with treatment response in the TME of solid tumors. It enables the systematic analysis of residual cancer populations after cisplatin treatment within the complex TME and further identification of predictive markers and targets for an individualized combination of chemotherapy with compounds targeting these mechanisms. The individual drug response of patients can be deeply evaluated and efficacious therapies can be further developed. Citation Format: Julia Thiel, Adrian Kneer, Weimeng Yu, Bernd Winkler, Georg Sauer, German Ott, Walter E. Aulitzky, Thomas E. Mürdter, Matthias Schwab, Chunguang Liang, Meng Dong. Evaluation of individual drug response in tumor microenvironment to cisplatin treatment in precision-cut tumor slices of ovarian cancer [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 639.

More detailed information on materials and methods

Legends for Supplementary Figures 1-8

The complex and dynamic microenvironment of tumors influences their development, progression, and response to therapy. In addition to cancer cells, solid tumors consist of a tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. A wide variety of secreted proteins maintain the heterotypic interactions between the cell types in the TME. However, it is not well understood how the TME responds to treatment at the proteome level. Here, we developed a unique nascent proteomic approach for precision-cut tumor slices to address this issue. Precision-cut tumor slices (PCTS) are a technology in which tumor tissues are cut to a defined thickness of 150-300 µm and cultured ex vivo for a certain time. PCTS maintain both the three-dimensional architecture and tumor heterogeneity and preserve their TME with respect to different cell types and the extracellular matrix. Our approach for PCTS nascent proteome analysis combines pulsed-SILAC (stable isotope labeling with amino acids in cell culture) with click chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon drug treatment. PCTS were generated from patient-derived xenografts and primary human ovarian tumors. After a depletion step, the PCTS were cultured in AHA-SILAC medium and treated with cisplatin. PCTS and culture media containing secreted proteins were harvested separately. Newly synthesized proteins were enriched via click chemistry and analyzed using mass spectrometry. A maximum labelling efficiency of >60% was achieved. Human PCTS showed a higher labelling efficiency than mouse xenografts. The PCTS of different tumors showed varying labeling efficiencies, indicating patient heterogeneity. Nascent proteome analysis enables the investigation of drug resistance and response in different patients. Cisplatin treatment resulted in the downregulation of >200 proteins in responsive tumors. A PCTS resistant to cisplatin did not show this response. Moreover, the corresponding patient had a worse clinical outcome than patients whose tumors were sensitive ex vivo. GSEA revealed the involvement of components such as cadherin binding and DNA translation. Protein-protein interactions can be predicted via STRING analysis. Tumor response or resistance was validated using viability assays and immunohistochemical staining for biomarkers of DNA damage and cell death. In conclusion, we established an ex vivo nascent proteome analysis method to study drug response within the complex TME, which can predict the tumor in vivo drug response. By combining the PCTS culture system with pulsed SILAC-AHA treatment, this approach allows the tracking of compositional and dynamic changes within the proteome and monitoring of the direct proteome response on a rapid timescale. It can be used to study cellular communication, predict therapeutic outcomes, and identify new therapeutic targets. Citation Format: Julia Thiel, Lina-Marie Wagner, Karim Aljakouch, Julia Schüler, Bernd Winkler, Kathrin Böpple, Thomas E. Mürdter, Georg Sauer, German Ott, Walter E. Aulitzky, Matthias Schwab, Jeroen Krijgsveld, Meng Dong. Nascent proteome analysis of drug response in precision-cut tumor slices [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 2020.

Table of TaqMan Assays (Applied Biosystems) used in this study

Supplemental Figures (Supplementary Fig. 1-8): Cell lines are HR proficient (Supplementary Fig. 1); Influence of hyperthermia on the quantity of intra-nuclear Pt-DNA adducts and intracellular doxorubicin concentration (Supplementary Fig. 2); Hyperthermia sensitizes tumor cells to drugs which induce PARylation without altering PARP of PARG protein levels (Supplementary Fig. 3); Hyperthermia leads to a delayed DNA strand break repair capacity in different cell lines from ovarian and colon cancer (Supplementary Fig. 4); HR competence in 14 of 15 samples from PC patients (Supplementary Fig. 5); Gamma-irradiation leads to induction of γH2AX foci in all cells independent of proliferation status (Supplementary Fig. 6); Hyperthermia has no acute effect on cisplatin or doxorubicin induced apoptosis (Supplementary Fig. 7); Combined treatment of cells with hyperthermia and PARPi slightly enhanced the potentiating effect of either treatment alone (Supplementary Fig. 8).

Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.

Abstract Background Cancer registries usually assess data of conventional treatments and/or patient survival. Beyond that, little is known about the influence of other predictors of treatment response related to the use of complementary therapies (CM) and lifestyle factors affecting patients’ quality and quantity of life. Methods INTREST is a prospective cohort study collecting register data at multiple German certified cancer centers, which provide individualized, integrative, in- and outpatient breast cancer care. Patient-reported outcomes and clinical cancer data of anticipated N = 715 women with pTNM stage I-III breast cancer are collected using standardized case report forms at the time of diagnosis, after completing neo−/adjuvant chemotherapy, after completing adjuvant therapy (with the exception of endocrine therapy) as well as 1, 2, 5, and 10 years after baseline. Endpoints for multivariable prediction models are quality of life, fatigue, treatment adherence, and progression-based outcomes/survival. Predictors include the study center, sociodemographic characteristics, histologic cancer and comorbidity data, performance status, stress perception, depression, anxiety, sleep quality, spirituality, social support, physical activity, diet behavior, type of conventional treatments, use of and belief in CM treatments, and participation in a clinical trial. Safety is recorded following the Common Terminology Criteria for Adverse Events. Discussion This trial is currently recruiting participants. Future analyses will allow to identify predictors of short- and long-term response to integrative breast cancer treatment in women, which, in turn, may improve cancer care as well as quality and quantity of life with cancer. Trial registration German Clinical Trial Register DRKS00014852 . Retrospectively registered at July 4th, 2018.

Purpose: Patients with estrogen receptor– and/or progesterone receptor–positive, early breast cancer benefit from hormonal treatment, yet high global death burdens due to high prevalence and long-term recurrence risk call for biomarkers to guide additional treatment approaches. Experimental Design: From a prospective, observational study of postmenopausal early breast cancer patients treated with tamoxifen or aromatase inhibitors, gene expression analyses of 612 tumors was performed using the NanoString Breast Cancer 360 panel to interrogate 23 breast cancer pathways. Candidate signatures associated with disease subtype and event-free survival (EFS) were obtained by cluster analysis, Cox modeling, and conditional inference trees, and were independently tested in 613 patients from BreastMark. Tumor-infiltrating lymphocytes (TIL) were assessed on tissue sections, and mutational burden was assessed in 36 tumors by whole-exome sequencing. Results: PAM50-derived classification distinguished lower-risk (Luminal A) from higher-risk subtypes (Luminal B, P = 0.04; HER2, P = 0.006; Basal, P = 0.008). In higher-risk patients, shorter EFS was associated with low androgen receptor [HR = 3.61; 95% confidence interval (CI), 1.72–7.56; P = 0.001] or high BRCAness signature expression (HR = 3.58; 95% CI, 1.19–10.7; P = 0.023). BRCAness was independently confirmed as a predictor of shorter EFS (HR = 2.64; 95% CI, 1.31–5.34; P = 0.007). About 13%–15% of patients, enriched for high-grade, higher-risk subtypes (P ≤ 0.0001), had strong expression of the Tumor Inflammation Signature (TIS) suggestive of an inhibited antitumor immune response. TIS scores were strongly associated with TIL numbers (P < 1e-30) but not with tumor mutation status. Conclusions: BRCA-related DNA repair deficiency and suppressed tumor immune responses may be clinically relevant predictors of endocrine therapy complementing treatment options in subgroups of hormone-sensitive early breast cancer.

Hintergrund Die minimal-invasive (laparoskopische) Operationstechnik wird als Alternative zum offenen Vorgehen (Laparotomie) in der Behandlung von Frauen mit frühem Zervixkarzinom angesehen, obgleich bisher keine belastbaren Studiendaten über dessen Einfluss auf die Prognose der Erkrankung vorliegen. Dieser Fragestellung haben sich gleich zwei Gruppen gewidmet: die einen in Form einer prospektiven Phase-III-Studie [1], die anderen in der einer retrospektiven Auswertung der amerikanischen National Cancer Database (NCDB) und der Surveillance, Epidemiology and End Results (SEER; [2]).

Abstract Although hyperthermia offers clinical appeal to sensitize cells to chemotherapy, this approach has been limited in terms of long-term outcome as well as economic and technical burden. Thus, a more detailed knowledge about how hyperthermia exerts its effects on chemotherapy may illuminate ways to improve the approach. Here, we asked whether hyperthermia alters the response to chemotherapy-induced DNA damage and whether this mechanism is involved in its sensitizing effect in BRCA-competent models of ovarian and colon cancer. Notably, we found that hyperthermia delayed the repair of DNA damage caused by cisplatin or doxorubicin, acting upstream of different repair pathways to block histone polyADP-ribosylation (PARylation), a known effect of chemotherapy. Furthermore, hyperthermia blocked this histone modification as efficiently as pharmacologic inhibitors of PARP (PARPi), producing comparable delay in DNA repair, induction of double-strand breaks (DSB), and cell cytotoxicity after chemotherapy. Mechanistic investigations indicated that inhibiting PARylation by either hyperthermia or PARPi induced lethal DSB upon chemotherapy treatment not only by reducing DNA repair but also by preventing replication fork slowing. Overall, our work reveals how PARP blockade, either by hyperthermia or small-molecule inhibition, can increase chemotherapy-induced damage in BRCA-competent cells. Cancer Res; 76(10); 2868–75. ©2016 AACR.

CD9 is the best-studied member of the tetraspanin family of transmembrane proteins. It is involved in various fundamental cellular processes and its altered expression is a characteristic of malignant cells of different origins. Despite numerous investigations confirming its fundamental role, the heterogeneity of CD9 or other tetraspanin proteins was considered only to be caused by posttranslational modification, rather than alternative splicing. Here we describe the first identification of CD9 transcript variants expressed by cell lines derived from fetal rat brain cells. Variant mRNA-B lacks a potential translation initiation codon in the alternative exon 1 and seems to be characteristic of the tumorigenic BT cell lines. In contrast, variant mRNA-C can be translated from a functional initiation codon located in its extended exon 2, and substantial amounts of this form detected in various tissues suggest a contribution to CD9 functions. From the alternative sequence of variant C, a different membrane topology (5 transmembrane domains) and a deviating spectrum of functions can be expected.