Chemosensitivity Studies Research Articles

A comparative study of carotid body chemosensitivity in lean and obese mice: Implication for obesity induced hypertension

Obesity is associated with increased circulating level of the hormone leptin. Previously we have shown in mice that, leptin induces hypertension acting on transient receptor potential melastatin 7 (Trpm7) channels in the carotid body (CB), a multi-modal peripheral chemoreceptor organ that controls breathing and drives sympathetic tone. Further, in mice leptin augments hypoxic ventilatory response which is abolished by CB denervation. This suggests a potential involvement of CB chemosensitivity. However, no comparative study of CB chemosensory responses has been conducted in lean and obese mice to support this notion. Based on these premises, we hypothesize that obese mice should have higher leptin induced CB chemosensory responses during normoxia and hypoxia compared to lean mice, and Trpm7 receptor antagonist should abolish/reduce these responses. To prove this, we measured afferent carotid sinus nerve (CSN) activity, using a novel ex vivo perfused mouse (male) CB preparation from lean (b.w.; 35 ± 0.5 gm) and diet induced obese (DIO) mice (b.w.; 53 ± 1.5gm) with C57BL/6J background. We found that compared to lean mice; (a) CSN response to hypoxia (PO2 =60 Torr) was 50% more in obese mice, (b) in presence of leptin (50nM), CSN responses to normoxia (PO2=100Torr) and hypoxia were augmented 50% and 150% more respectively in obese mice. Finally, leptin induced increase in CSN responses were partially reduced by Trpm7 antagonist FTY720 (3μM) in obese compared to lean mice. In conclusion, CB chemosensitivity is enhanced in obese compared to lean mice and CB Trpm7 channel could be a therapeutic target for obesity related hypertension. Grant support: NIH R01 HL1331000. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cytotoxicity of Ruthenium(II) Arene Complexes Containing Functionalized Ferrocenyl β-Diketonate Ligands.

The synthesis and characterization of 24 ruthenium(II) arene complexes of the type [(p-cym)RuCl(Fc-acac)] (where p-cym = p-cymene and Fc-acac = functionalized ferrocenyl β-diketonate ligands) are reported, including single-crystal X-ray diffraction for 21 new complexes. Chemosensitivity studies have been conducted against human pancreatic carcinoma (MIA PaCa-2), human colorectal adenocarcinoma p53-wildtype (HCT116 p53+/+) and normal human retinal epithelial cell lines (APRE-19). The most active complex, which contains a 2-furan-substituted ligand (4), is 5x more cytotoxic than the analogs 3-furan complex (5) against MIA PaCa-2. Several complexes were screened under hypoxic conditions and at shorter-time incubations, and their ability to damage DNA was determined by the comet assay. Compounds were also screened for their potential to inhibit the growth of both bacterial and fungal strains.

Role of Intracellular and Extracellular Annexin A1 in MIA PaCa-2 Spheroids Formation and Drug Sensitivity.

Simple SummaryIn order to improve the investigation of pancreatic cancer (PC), often supported through analyzes two-dimensional (2D) cell monolayers, we proposed to create a spheroid-based in vitro three-dimensional (3D) model using wild-type (WT) and ANXA1 knock-out (KO) MIA PaCa-2 PC cells. However, the production of spheroids still represents a technical challenge. Here, we have developed a protocol to obtain well-organized spheroids and have proved that Annexin A1 (ANXA1) affects the spheroid formation, because the WT cells have a greater ability to form this 3D model when compared to the ANXA1 KO examples. We also investigated how ANXA1 action could influence the PC pharmacological response both in basal conditions and by mimicking a tumor system through the addition of autocrine EVs. ANXA1, via EVs, significantly improves the formation, the stability and the drug resistance of this model, particularly compared to the ANXA1 KO one, which shows a structural instability and a greater drug sensitivity.Among solid tumors, pancreatic cancer (PC) remains a leading cause of death. In PC, the protein ANXA1 has been identified as an oncogenic factor acting in an autocrine/paracrine way, and also as a component of tumor-deriving extracellular vesicles. Here, we proposed the experimental protocol to obtain spheroids from the two cell lines, wild-type (WT) and Annexin A1 (ANXA1) knock-out (KO) MIA PaCa-2, this last previously obtained through CRISPR/Cas9 genome editing system. The use of three-dimensional (3D) models, like spheroids, can be useful to mimic tumor characteristics and for preclinical chemo-sensitivity studies. By using PC spheroids, we have assessed the activity of intracellular and extracellular ANXA1. Indeed, we have proved that the intracellular protein influences in vitro tumor development and growth by spheroids analysis, in addition to defining the modification about cell protein pattern in ANXA1 KO model compared to the WT one. Moreover, we have tested the response to FOLFIRINOX chemotherapy regimen whose cytostatic effect appeared notably increased in ANXA1 KO spheroids. Additionally, this study has highlighted that the extracellular ANXA1 action is strengthened through the EVs supporting spheroids growth and resistance to drug treatment, mainly affecting tumor progression. Thus, our data interestingly suggest the relevance of ANXA1 as a potential therapeutic PC marker.

Dynamics of Serum Thymidine Kinase 1 at the First Cycle of Neoadjuvant Chemotherapy Predicts Outcome of Disease in Estrogen-Receptor-Positive Breast Cancer.

Simple SummaryChemotherapy before surgery (NAC) is an option for high-risk breast cancer (BC) patients. Pathologic complete response (pCR) predicts long-term outcome and has become a surrogate biomarker for survival. pCR is, however, reached in only <10% of hormone-receptor-positive (ER+) patients and is of limited prognostic value. Biomarkers able to predict outcome early during NAC would facilitate individualized therapy with the possibility to adjust or interrupt an ineffective therapy. Here, it is shown that differential response of the serum concentration of thymidine kinase 1, an enzyme involved in the DNA synthesis and released from the tumor into the blood, 48 h after the first cycle of NAC, predicts long-term outcome in localized advanced ER+/HER2-BC. The different reactions to chemotherapy could be used to guide this process early during NAC and utilized to identify mechanisms of tumor sensitivity that could provide a prediction of long-term outcome prior to chemotherapy.Pathologic complete response (pCR) predicts the long-term outcome of neoadjuvantly treated (NAC) breast cancer (BC) but is reached in <10% of hormone-receptor-positive patients. Biomarkers enabling adjustment or interruption of an ineffective therapy are desired. Here, we evaluated whether changes in the serum concentration of thymidine kinase 1 (sTK1) during NAC could be utilized as a biomarker. In the PROMIX trial, women with localized HER2- BC received neoadjuvant epirubicin/docetaxel in six cycles. sTK1 was measured with an ELISA in 54 patients at cycles 1–4 and in an additional 77 patients before and 48 h after treatment 1. Treatment resulted in a 2-fold increase of sTK1 before and a 3-fold increase 48 h after the cycles, except for the first cycle, where half of the patients reacted with a significant decrease and the other half with an increase of sTK1. In Kaplan–Meier estimates of ER+ patients divided by the median of the post/pre-treatment sTK1 ratio at the first treatment cycle, OS was 97.7% and 78% (p = 0.005), and DFS was 90.7% and 68% (p = 0.006), respectively. Thus, the response of sTK1 at the first cycle of chemotherapy could be used both as an early biomarker for the guidance of chemotherapy and for the study of inherent tumor chemo-sensitivity, which could predict long-term outcome prior to therapy.

Chemosensitivity analysis and study of gene resistance on tumors and cancer stem cell isolates from patients with colorectal cancer.

Colorectal cancer (CRC) is one of the main causes of mortality. Recent studies suggest that cancer stem cells (CSCs) can survive after chemotherapy and promote tumor invasiveness and aggression. According to a higher hierarchy complexity of CSC, different protocols for isolation, expansion, and characterization have been used; however, there are no available resistance biomarkers that allow predicting the clinical response of treatment 5-fluorouracil (5FU) and oxaliplatin. Therefore, the primary aim of the present study was to analyze the expression of gene resistance on tumors and CSC-derived isolates from patients CRC. In the present study, adenocarcinomas of the colon and rectum (CRAC) were classified based on an in vitro adenosine triphosphate-based chemotherapy response assay, as sensitive and resistant and the percentage of CD24 and CD44 markers are evaluated by immunohistochemistry. To isolate resistant colon-CSC, adenocarcinoma tissues resistant to 5FU and oxaliplatin were evaluated. Finally, all samples were sequenced using a custom assay with chemoresistance-associated genes to find a candidate gene on resistance colon-CSC. Results showed that 59% of the CRC tissue analyzed was resistant and had a higher percentage of CD44 and CD24 markers. An association was found in the expression of some genes between the tumor-resistant tissue and CSC. Overall, isolates of the CSC population CD44+ resistant to 5FU and oxaliplatin demonstrated different expression profiles; however, the present study was able to identify overexpression of the KRT-18 gene, in most of the isolates. In conclusion, the results of the present study showed overexpression of KRT-18 in CD44+ cells is associated with chemoresistance to 5FU and oxaliplatin in CRAC.

Establishment and Characterization of a Novel Dedifferentiated Chondrosarcoma Cell Line DDCS2.

BackgroundThe dedifferentiated variant of chondrosarcoma is highly aggressive and carries an especially grim prognosis. While chemotherapeutics has failed to benefit patients with dedifferentiated chondrosarcoma significantly, preclinical chemosensitivity studies have been limited by a scarcity of available cell lines. There is, therefore, an urgent need to expand the pool of available cell lines.MethodsWe report the establishment of a novel dedifferentiated chondrosarcoma cell line DDCS2, which we isolated from the primary tumor specimen of a 60-year-old male patient. We characterized its short tandem repeat (STR) DNA profile, growth potential, antigenic markers, chemosensitivity, and oncogenic spheroid and colony-forming capacity.ResultsDDCS2 showed a spindle to polygonal shape and an approximate 60-hour doubling time. STR DNA profiling revealed a unique genomic identity not matching any existing cancer cell lines within the ATCC, JCRB, or DSMZ databases. There was no detectable contamination with another cell type. Western blot and immunofluorescence assays were consistent with a mesenchymal origin, and our MTT assay revealed relative resistance to conventional chemotherapeutics, which is typical of a dedifferentiated chondrosarcoma. Under ex vivo three-dimensional (3D) culture conditions, the DDCS2 cells produced spheroid patterns similar to the well-established CS-1 and SW1353 chondrosarcoma cell lines.ConclusionOur findings confirm DDCS2 is a novel model for dedifferentiated chondrosarcoma and therefore adds to the limited pool of current cell lines urgently needed to investigate the chemoresistance within this deadly cancer.

The bone marrow stroma capacity to confer resistance to leukemia cells from Ara-C treatment as a prognostic factor for overall survival in AML.

e18500 Background: The interaction of acute myeloid leukemia (AML) with the bone marrow stroma (BMS) determines a protective microenvironment that favors leukemia development and resistance to chemotherapy. We developed an in vitro platform to study leukemia and stroma interaction, and showed that BMS secretes soluble factors protecting AML cells from Ara-C-induced apoptosis, which correlated with clinical patient outcomes. Methods: BMS from AML patients and healthy donors were cultured to perform chemo-sensitivity studies with Ara-C on THP-1 cells. A Resistance Factor was calculated (RF = IC50 stroma conditioned medium (CM)/IC50 control medium) to classify BMS as “protective” (PS: confers chemo-resistance) or “non-protective” (NPS: does not confer chemo-resistance). Characterization of myofibroblasts in BMS cultures was performed by WB. Quantification of cytokines from primary BMS CM was performed by Luminex. The differential expression of epithelial-mesenquimal-like (EMT-like) and stem cell-markers in THP-1 cells incubated with primary BMS CM was measured by qPCR. Results: We recruited 90 AML patients and 10 healthy BM donors. AML cohort showed 31 patients with PS and 59 patients with NPS. PS patients had a significant poor overall survival (OS) compared with NPS patients (44% versus 70% OS in 2.5 years, median survival 7.3 months versus 20.7 months, HR 2.36). The stroma from healthy BM donors did not confer resistance to THP-1 cells. BMS in both, PS and NPS have activated myofibroblasts and analysis of cytokine expression showed differential expression between groups. THP-1 cells incubated with PS showed significant expression of EMT- and stem cell-markers like Twist, Vimentin, CD44 and CD34 compared to THP-1 cells incubated with NPS and control medium. Conclusions: The capacity of the BMS from AML patients to modulate chemoresistance is a strong prognostic factor for OS, and PS patients have a worst OS compared with NPS patients. Stroma from healthy donors has a NPS phenotype. We propose that the leukemia is capable of educating the stroma to acquire secondarily the capacity to confer resistance to leukemia cells to favor tumor progression and chemoresistance.

Ethical issues in cancer care through ayurveda

The second most common disease in India responsible for maximum mortality is cancer, with an incidence of about 0.3 million deaths per year. Although there are good diagnostic methods and treatment, cancer is still a big risk to our society. Ayurveda is the most commonly used complementary and alternative medicine in India, as it is add-on to conventional cancer care. Now, this is need of the hour to make collaboration between Ayurveda and modern physicians with mutual trust in the management of cancer. In various meetings, standard treatment protocols have been set, and integrative oncology programs have been initiated in a few institutes across India. But still, there are some ethical issues regarding this integrative oncology. Today is the era of evidence-based medicine, and we are lacking in generating evidence of the benefits of Ayurveda intervention. Also, there is a deficit of publications in systematic reviews of prior research studies, exploring the safety and efficacy of Ayurveda in the management of cancer, ensuring comprehensive clinical documentation based on categorization of patients. We have enough knowledge about the mode of action of herbal as well as modern drugs, but we are not aware of herb–modern drug interaction. Hence, herb–modern drug interaction studies and chemosensitivity studies, based on both laboratory research and clinical observations, should be conducted. Acknowledgment of the medicolegal and ethical issues involved in integrative oncology and developing strategies for clinical practice and research should be made. Making available insurance coverage for integrative oncology care, keeping in mind the aim of universal health coverage, will facilitate more number of patients toward integrative management for cancer. Through appropriate cooperation with modern medicine, Ayurveda can make substantial contributions for the advances of integrative oncology. This can discover the specific health challenges and open up new opportunities for the development of integrative Ayurveda oncology in the world.

Synthesis, characterization and chemosensitivity studies of half-sandwich ruthenium, rhodium and iridium complexes containing к1(S) and к2(N,S) aroylthiourea ligands

The reaction of [(p-cymene)RuCl2]2 and [Cp*MCl2]2 (M = Rh/Ir) metal precursors with aroylthiourea ligands (L1-L3) yielded a series of neutral mono-dentate complexes 1–9. The neutral mono-dentate coordination of aroylthiourea with metals via S atom was confirmed by single crystal X-ray diffraction study. Further reaction of mono-dentate complexes 1–9 with excess NaN3 in polar solvent resulted in the formation of highly strained four member ring к2(N,S) azido complexes 10–18. Further these complexes were treated with activated alkynes to isolate triazole complexes, but unfortunately the reaction was unsuccessful. All these complexes were fully characterized by various spectroscopic techniques. The molecular structures of the representative complexes have been determined by single crystal X-ray diffraction studies. The molecular structures of the complexes revealed typical piano stool geometry around the metal center. The chemosensitivity activities of the complexes 1–9 evaluated against the cancer cell line HCT-116 (human colorectal carcinoma) and ARPE-19 (human retinal epithelial cells) cell line. Of these, complex 3 was the most potent and whilst its potency was less than cisplatin, its selectivity for cancer as opposed to non-cancer cell lines in vitro was comparable to cisplatin.

Stevioside mediated chemosensitization studies and cytotoxicity assay on breast cancer cell lines MDA-MB-231 and SKBR3.

Cancer is one of the most impacting life-threatening disease for the human populace. Hence, over the years we have seen a consistent interest to study and investigate new treatments to cure and prevent this disease. Medicinal plants have played a progressive part in treatment since many years. In this research study, we have explored the cytotoxicity effect of purified bioactive compound isolated from Stevia rebaudiana leaves and the key mechanism responsible for apoptosis in human breast cancer cells. The anticancer properties of Stevia rebaudiana leaves has been suggested in earlier literature. Hence, the aim of this study was to investigate the cytotoxicity of purified stevioside in human breast cancer cell lines MDA-MB-231 and SKBR3. Results showed that purified stevioside inhibited the growth of cancerous cell lines. The IC50 obtained after treatment with stevioside on cancer cells MDA-MB-231 and SKBR3 are 55 µM and 66 µM respectively. This shows purified stevioside is capable of inducing apoptosis indicating its promising anticancer activity. However, so far chemosensitization effects of stevioside on breast cancer have not been fully explained by other studies. Hence, additionally, this study also evaluates the chemosensitization potential of stevioside in combination with 5-FU. This research study shows the importance of Stevia rebaudiana as a good source of bioactive compounds with high anti-cancer property.

Highlights of This Issue

Patient-derived xenograft (PDX) received much attention as a mouse model for chemosensitivity studies. However, its practical problems have also been highlighted recently (e.g., Nature 560:156-157, 2018). By transplanting cultured tumor-initiating cells (cancer stem cells) into nude mice, Maekawa, Miyoshi and colleagues show that some of such key problems can be overcome, and that the chemosensitivity in this PDSX (patient-derived spheroid xenograft) model precisely reflects the findings in a retrospective study with the colorectal cancer patients. These results suggest that PDSX could be an alternative model to PDX with improved efficiency that can help the development of personalized clinical service.Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy remains challenging. CRPC is driven by aberrant activation of androgen receptor (AR) through different mechanisms, including expression of splice variants (e.g., AR-V7). Herein, Singh and colleagues present in vitro and in vivo evidence for therapeutic vulnerability of prostate cancer cells, including those that are resistant to enzalutamide, to a novel plant-based small molecule (leelamine).Long term control of metastatic colorectal cancer (CRC) remains a major unmet medical need worldwide. In this article, Wu and colleagues report a novel tetravalent T cell engaging bispecific antibody (T-BsAb) called huA33-BsAb which targets CRC associated antigen GPA33. The in vivo subcutaneous and intraperitoneal xenograft models demonstrate that HuA33-BsAb drives circulating polyclonal T cells to infiltrate and to kill GPA33 positive colon and gastric cancer cells, including cancers with high risk genotypes. Using a novel affinity maturation system for BsAb, the authors found that picomolar anti-GPA33 affinity did not appreciably improve the antitumor properties of huA33-BsAb.Epithelial to mesenchymal transition (EMT) is one of the mechanisms of acquired resistance to EGFR-TKIs in lung cancers with EGFR mutations. In this study, Suda and colleagues identified that high CD44 expression is correlated with mesenchymal phenotype in TKI-resistant cell lines and tumors. In addition, the authors found cell lines that often acquire resistance via EMT (HCC4006 and NCI-H1975 cells) have high CD44 expression prior to EGFR-TKI exposure. These findings suggest that CD44 is a useful biomarker to predict the emergence of EMT-mediated resistance to EGFR-TKIs and can be a therapeutic target to prevent EMT-mediated resistance.

A Chemosensitivity Study of Colorectal Cancer Using Xenografts of Patient-Derived Tumor-Initiating Cells.

Current genomic and gene expression analyses provide versatile tools to improve cancer chemotherapy. However, it is still difficult to predict whether each patient responds to a particular regimen or not. To predict chemosensitivity in each patient with colorectal cancer, we developed an evaluation method using the primary tumor-initiating cells (TIC, aka cancer stem cells) xenografted in nude mice subcutaneously (patient-derived spheroid xenografts; PDSX). Simultaneously, we also prepared the conventional patient-derived xenografts (PDX) from the same patients' tumors and compared the dosing results with those of PDSXs. We further compared the chemosensitivities of PDSXs with those of 7 patients who had been given regimens such as FOLFOX and FOLFIRI to treat their metastatic lesions. As per the results, the PDSX method provided much more precise and predictable tumor growth with less variance than conventional PDX, although both retained the epithelial characteristics of the primary tumors. Likewise, drug-dosing tests showed essentially the same results in PDXs and PDSXs, with stronger statistical power in PDSXs. Notably, the cancer chemosensitivity in each patient was precisely reflected in that of the PDSX mice along the clinical course until the resistance emerged at the terminal stage. This "paraclinical" xenograft trials using PDSXs may help selection of chemotherapy regimens efficacious for each patient, and, more importantly, avoiding inefficient ones by which the patient can lose precious time and QOL. Furthermore, the PDSX method may be employed for evaluations of off-label uses of cancer chemotherapeutics and compassionate uses of yet-unapproved new drugs in personalized therapies. Mol Cancer Ther; 17(10); 2187-96. ©2018 AACR.

Synthesis, structural and chemosensitivity studies of arene d6 metal complexes having N‐phenyl‐N´‐(pyridyl/pyrimidyl)thiourea derivatives

The d6 metal complexes of thiourea derivatives were synthesized to investigate its cytotoxicity. Treatment of various N‐phenyl‐N´ pyridyl/pyrimidyl thiourea ligands with half‐sandwich d6 metal precursors yielded a series of cationic complexes. Reactions of ligand (L1‐L3) with [(p‐cymene)RuCl2]2 and [Cp*MCl2]2 (M = Rh/Ir) led to the formation of a series of cationic complexes bearing general formula [(arene)M(L1)к2(N,S)Cl]+, [(arene)M(L2)к2(N,S)Cl]+ and [(arene)M(L3)к2(N,S)Cl]+ [arene = p‐cymene, M = Ru (1, 4, 7); Cp*, M = Rh (2, 5, 8); Cp*, Ir (3, 6, 9)]. These compounds were isolated as their chloride salts. X‐ray crystallographic studies of the complexes revealed the coordination of the ligands to the metal in a bidentate chelating N,S‐ manner. Further the cytotoxicity studies of the thiourea derivatives and its complexes evaluated against HCT‐116 (human colorectal cancer), MIA‐PaCa‐2 (human pancreatic cancer) and ARPE‐19 (non‐cancer retinal epithelium) cancer cell lines showed that the thiourea ligands displayed no activity. Upon complexation however, the metal compounds possesses cytotoxicity and whilst potency is less than cisplatin, several complexes exhibited greater selectivity for HCT‐116 or MIA‐PaCa‐2 cells compared to ARPE‐19 cells than cisplatin in vitro. Rhodium complexes of thiourea derivatives were found to be more potent as compared to ruthenium and iridium complexes.

Comparative study of chemo-sensitivity expressed as micronuclei in lymphocytes of breast cancer patients, their unaffected first degree relatives and normal controls as a possible prognostic marker

Comparative study of chemo-sensitivity expressed as micronuclei in lymphocytes of breast cancer patients, their unaffected first degree relatives and normal controls as a possible prognostic marker

Abstract 4943: Orthotopic transplantation of pancreatic cancer PDX models increases murine stroma content, but does not influence therapeutic response to standard of care

Abstract Background: Reasons for the poor prognosis of pancreatic cancer are advanced and inoperable tumor stages at time of diagnosis and resistance to conventional therapies. Patient-derived xenografts (PDX) represent a valuable tool for the prediction of therapy response, the identification of new biomarkers and therapeutic targets or pancreatic cancer specific activated pathways (MAPK, hedgehog). However, PDX tumors differ from patient tumors as their surrounding tissue is replaced by murine stroma within 3 to 9 weeks after primary transplantation. Since the desmoplastic stroma has an impact on the progression and treatment of pancreatic cancer, we investigated the characteristics and function of murine stroma components in PDX models of pancreatic ductal adenocarcinoma (PDAC). Methods: We analyzed the relevance of murine cancer-associated fibroblasts (CAFs) for therapeutic response by comparing orthotopic with s.c. transplantation in PDX. We selected a cohort of 3 PDAC PDX with different growth rates (slow, median and fast growth). Human pancreatic tumor material was implanted s.c. or orthotopically into immunodeficient mice. After successful engraftment, the chemosensitivity to standard of care (SoC) drugs (focus on 2 to 3 drugs) was determined according to clinically relevant and optimized schedules. Growth of orthotopic PDX models was monitored via high-resolution ultrasound. Cryo- and formalin-preserved tumor tissues from the chemosensitivity studies were collected and analyzed for specific stroma markers (collagen I and αSMA). Results: In general, the response profiles in our experiments have closely reflected patient’s response in the clinic for the drug combination gemcitabine and nab-paclitaxel. There were no differences in response when comparing s.c. with orthotopic growing tumors. Semi-quantitative analysis of tumor sections showed that orthotopic tumors contain more murine stroma compared to s.c. tumors. Comparison of treated and untreated tumors revealed a strong reduction of the tumor cell content by the SoC treatment, whereas CAFs (collagen I and αSMA positive fraction) were not affected. Conclusion: In summary, this study revealed that orthotopic transplantation results in the growth of tumors with an improved and functional tumor microenvironment. Therapy is effective on the pancreatic cancer cells, however, tumor stroma seems apparently to be inherent resistant towards conventional therapy. The remaining tumor stroma might provide a survival nice for some tumor cells - leading to the frequently observed disease relapse. Therefore, orthotopic PDX are considered to be the more clinical relevant models in comparison to s.c. PDX because they mimic closely the human pancreatic cancer microenvironment and also favor the development of metastasis. Further, targeting the tumor stroma should be implicated into clinical treatment strategies. Citation Format: Ulrike Pfohl, Diana Behrens, Iduna Fichtner, Jens Hoffmann, Britta Büttner, Wolfgang Walther. Orthotopic transplantation of pancreatic cancer PDX models increases murine stroma content, but does not influence therapeutic response to standard of care [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2017-4943

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Abstract 4080: Analysis of murine stromal components in patient-derived xenograft (PDX) models of pancreatic cancer

Abstract Pancreatic cancer remains a lethal disease with only 3 - 8% of patients surviving 5 years after initial diagnosis (WHO, 2012). Reasons for this poor situation are advanced and inoperable tumor stages at time of diagnosis and resistance to conventional therapies. One bottleneck in the development of novel therapies is the restricted availability of preclinical models of high clinical relevance. Since the desmoplastic stroma has impact on the progression and treatment of pancreatic cancer, we investigated the attributes of the murine stroma in patient-derived xenografts that completely replaced the human surrounding tissue within a few months after primary transplantation. We elucidated the functionality of murine tumor microenvironment for growth and therapeutic response in a cohort of well-characterized pancreatic cancer (PDAC) PDX. PDX are a valuable tool for the prediction of therapy response, the identification of new biomarkers and therapeutic targets or pancreatic cancer specific pathways. In this study, 57 patient tumors were collected and immediately transplanted into immunodeficient mice. So far, 14 out of 57 samples were established as passageable pancreatic cancer xenografts (PDX). All engrafted PDX are poorly or moderately differentiated adenocarcinomas. Global gene expression analysis and determination of cancer associated mutations revealed K-ras mutations in 13 and additionally p53 mutations in 9 out of 14 PDX. Furthermore, chemosensitivity to standard of care (SoC) drugs was determined by using clinically relevant and optimized schedules and doses. The testing revealed that the response to Gemcitabine (1/10 responder) was moderate within the PDX panel, while the most efficient drug was Abraxane with 5 out of 10 responders. In general, the response profile of all PDX closely reflected patient's situation in the clinic. Cryo- and formalin-preserved tumor tissues of these chemosensitivity studies were investigated for markers of desmoplastic stroma (SPARC, alpha-SMA, FAP and collagen I). Immunohistochemistry and real-time PCR revealed, that even the replacing murine stroma is characterized by a distinct reactivate nature. Semi-quantitative analysis of stromal components showed that the tumor surrounding tissue mass was not significantly reduced due to therapeutic intervention. Though the tumor burden was diminished under SoC, the mRNA expression level of SPARC and FAP was unaffected in corresponding samples of the treatment groups compared to vehicle-treated control. The same effect was found for alpha-SMA and collagen I in immunohistochemically stained specimens. In summary, this study revealed a functional tumor environment of murine origin in patient-derived xenografts of pancreatic cancer and furthermore an apparently inherent resistance of this stromal tissue towards conventional therapy. Thus, targeting the tumor microenvironment should be implicated into clinical decisions. Citation Format: Diana Behrens, Ulrike Pfohl, Britta Büttner, Jens Hoffmann, Wolfgang Walther, Iduna Fichtner. Analysis of murine stromal components in patient-derived xenograft (PDX) models of pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4080.

Establishment and characterization of a penile cancer cell line, penl1, with a deleterious TP53 mutation as a paradigm of HPV-negative penile carcinogenesis.

PurposeTo establish penile cancer (PeCa) cell lines for the study of molecular mechanisms of carcinogenesis and testing therapeutic reagents.Materials and MethodsWe successfully established two PeCa cell lines from fresh tumor tissues from 21 cases. One cell line named Penl1 was isolated from a lymph node metastasis (LNM) of penile squamous cell carcinoma (PeSCC), usual type and comprehensively characterized here. Our in-depth characterization analysis of the Penl1 cell line included morphology, tumorigenicity, genetic characteristics, protein expression, biology, and chemosensitivity. Penl1 was authenticated by single tandem repeat (STR) DNA typing.ResultsComparative histomorphology, genetic characteristics, and protein expression patterns revealed essential similarities between the cell line and its corresponding LNM. In-depth characterization analysis of Penl1 cell line revealed tumorigenicity in immunodeficient mice, negative human papilloma virus (HPV) and mycoplasma infection, TP53 mutations and sensitivity to cisplatin and epirubicin. STR DNA typing did not match any cell lines within three international cell banks. The limitation of this study is that one patient cannot represent the complete heterogeneity of PeCa, especially primary tumor.ConclusionsWe established and characterized an HPV-negative and moderately differentiated PeCa cell model with a TP53 missense mutation from a PeSCC, usual type patient. A preliminarily study of carcinogenesis and chemosensitivity suggests that this cell model carries a tumor suppressor gene mutation and is sensitive to chemotherapy drugs.

Final Results of the Phase I/II Study of Chemosensitization Using the CXCR4 Inhibitor Plerixafor in Combination with Bortezomib in Patients with Relapsed or Relapsed/Refractory Multiple Myeloma

PURPOSE: This study aimed to determine activity and safety of the CXCR4 inhibitor plerixafor in combination with bortezomib and dexamethasone in patients with relapsed or refractory Multiple Myeloma (MM). This was based on our preclinical studies showing that plerixafor (Mozobil, Sanofi Corporation) induces de-adhesion of MM cells and sensitization to bortezomib in preclinical animal models.PATIENTS AND METHODS: Theprimary endpoint of the phase I study was the maximum tolerated dose (MTD) and for the phase II study, the safety and response rate of the combination. Eligibility criteria included patients with relapsed or relapsed/refractory MM with 1-5 prior lines of therapy including bortezomib (unless patients were refractory to bortezomib). The phase I included 8 cohorts with different doses and two treatment schedules. In cohorts 1-5, patients received plerixafor at the recommended dose sq on days 1-6 of each cycle and bortezomib at the recommended dose twice a week on days 3, 6, 10, and 13 every 21 days. In cohort 5b-6, plerixafor was given at the recommended dose sq on days 1, 3, 6, 10, and 13 and bortezomib was given at the recommended dose twice a week on days 3, 6, 10, and 13 every 21 days. For the phase II portion patients received plerixafor at the MTD established in phase I of trial, 320 mcg/kg sq on days 1, 2, 3, 6, 10, and 13. Bortezomib was given 1.3 mg/m2 IV or sq twice a week on days 3, 6, 10, 13, every 21 days. Dexamethasone was given at 40mg on days of Bortezomib.RESULTS: A total of 58 patients were enrolled on this study from June 2009 to March 2015, with 25 on the phase I and 33 on the phase II study. In the phase I study, the median age was 60 years (range, 43-85), the median number of prior therapies was 2 (range, 1-4), with all but 3 patients receiving prior bortezomib. The median number of cycles on therapy was 4 (1-12). Dose limiting toxicities including insomnia, restlessness, and psychosis were observed in two patients at dose level 6 (plerixafor 0.40 mg/kg and bortezomib 1.3 mg/m2). Therefore, 3 additional patients were enrolled at dose level 5b (plerixafor 0.32 mg/kg and bortezomib 1.3 mg/m2). There were no grade 4 toxicities. Grade 3 toxicities included lymphopenia (40%), hypophosphatemia (20%), anemia (10%), hyponatremia (10%), hypercalcemia (10%), and bone fracture due to myeloma bone disease (10%). Twenty-three patients were evaluable for response, including 1 (4%) complete response (CR), 1 (4%) very good partial response (VGPR), 1 partial remission (PR) and 2 (9%) MR, and 15 (65%) having stable disease with only 3 (13%) progressive disease (PD). In the phase II study, the median age was 63 (46-83). The median number of prior therapies was 2 (1-5), with 22 (66%) who have received prior bortezomib. The median number of cycles on therapy is 5 (1-24). The response rate included 5 VGPR (16%), 11 PR (35%) with an overall response rate of 51% and another 11 (35%) stable disease. Grade 3/4 toxicities included thrombocytopenia (68%), lymphopenia (6%), hypophosphatemia (2%), anemia (4%), infections (4%), hyponatremia (2%), hypercalcemia (2%) and neurological toxicity (2%). We also examined in vivo mobilization of plasma cells, CD34+ hematopoietic stem cells and other accessory bone marrow cells. Analysis of these samples showed rapid mobilization of plasma cells at 2 hours post-plerixafor with a rapid return to normal levels at 4 and 24 hours post plerixafor. CONCLUSIONS: The combination of plerixafor and bortezomib is generally well tolerated with minimal neuropathy or other toxicities seen to date. The responses observed are strongly encouraging with 51% ORR in this relapsed and refractory population. This study was supported by R01CA133799-01, and by Sanofi and Takeda Corporations. DisclosuresOff Label Use: Plerixafor in myeloma. Azab:Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner . Schlossman:Millennium: Consultancy. Richardson:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees.

ATCT-14CHEMOSENSITIVITY OF PRIMARY AND METASTATIC CNS TUMORS TO 4-DEMETHYL-4-CHOLESTERYLOXYCARBONYLPENCLOMEDINE (DM-CHOC-PEN) AND TEMOZOLOMIDE

BACKGROUND: The efficacy of TMZ in the treatment of high-grade astrocytomas is limited by drug-resistance mediated by O6-methylguanine-DNA methyltransferase (MGMT). Brain metastases are difficult to treat and currently no standard chemotherapeutic therapies exist. 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a polychlorinated pyridine cholesteryl carbonate whose mechanism of action is via alkylation of DNA at N7-guanine. This agent has had early therapeutic success in treating both primary and metastatic CNS malignancies in Phase I and Phase II clinical trials. METHODS: To better understand the sensitivity and mechanisms of resistance in primary and metastatic CNS tumors, we collected tissue from subjects (n = 38) enrolled between March 2014 - January 2015 for chemosensitivity studies. These included high-grade astrocytomas (9), metastatic brain tumors (6) meningiomas (6), and other more benign tumors such as pituitary adenomas (4), hemangioblastoma (1), and chondrosarcoma (1). Using viable tumor cell explants from surgery, cells were grown under standard conditions (RPMI/FBS, 5%CO2, 37°C), in vitro chemosensitivity profiles were obtained comparing DM-CHOC-PEN vs. TMZ and other commonly used anti-cancer agents. RESULTS: Tumor cells exhibited higher susceptibility to DM-CHOC-PEN than to TMZ (RR = 3.19, p = 0.0029, 95% confidence interval [CI], 1.3-7.8 vs. TMZ). In a subgroup analysis of high-grade astrocytomas, atypical meningiomas, and secondary metastases in this cohort, an association was noted between TMZ-resistance (µIC50 = 3.3, S.D. = 0.46) and DM-CHOC-PEN-sensitivity (µIC50 = 1.0, S.D = 0.26) using two-sample independent, Student's t-test with Fisher's exact P-value (p = 0.0001, t(23.72) = 4.5, df = 27.17). CONCLUSIONS: These preliminary results suggest that DM-CHOC-PEN may promote cell death in tumors possessing acquired MGMT-mediated drug-resistance. Furthermore, it supports roles for this promising new agent in the treatment of primary and metastatic brain tumors and in treatment of high-grade astrocytomas, either in combination with TMZ or alone in cases of TMZ-resistance.