Efficient, aggregation-free extracellular vesicles (EVs) labeling is essential for studying their dynamics in-vitro and in-vivo . However, traditional dyes introduce limitations including aggregation, membrane intercalation, fluorescence transfer and inconsistent performance across EV sources thus distorting quantification, altering surface properties and confounding uptake and biodistribution analyses. Here, we systematically evaluated CytoLight, a luminal dye traditionally used for live-cell imaging, as an alternative for EV quantification, characterization, uptake analysis and in-vivo tracking, benchmarking it against PKH26, CFSE and ExoBrite across multiple platforms. CytoLight generated stable, intravesicular fluorescence without aggregation or membrane alteration, eliminating artifacts characteristic of conventional dyes. Using fluorescence-NTA and single-EV flow cytometry, CytoLight showed more consistent labeling across EV types than CFSE or ExoBrite, while avoiding PKH-related micelle-driven artifacts and exhibited compatibility with CD81 dual-detection. In uptake assays, CytoLight produced EV-specific endocytosis-dependent internalization signals exceeding labeled-BPS/protein controls. In-vivo , CytoLight-labeled EVs enabled fluorescent biodistribution mapping showing conventional EV tropism patterns distinguishable from labeled-PBS controls. These findings establish CytoLight as an effective, aggregation-free EV-labeling strategy. Its stability, specificity, compatibility with single-EV platforms and reliable performance in both cellular uptake and biodistribution studies position CytoLight as a practical, scalable alternative to current dyes, providing a stronger foundation for standardized and reproducible EV research. ### Competing Interest Statement The authors have declared no competing interest. The Varda and Boaz Dotan Research Center in Hemato-Oncology Grant Alan Fenton grant for research in FMRC

The arginine‐glycine‐glutamic acid (RGD) sequence, an αvβ3 integrin recognition site, is overexpressed in malignancies and neovasculature, making it a potential therapeutic target. Herein, efficacy/safety of tumor‐targeted RGD‐based proteinoid nanocapsules (NCs) entrapping a synergistic combination of two drugs—palbociclib (Pal), a CDK4/6 inhibitor, and alpelisib (Alp), a P13K inhibitor, as a cancer treatment, is assessed. P(RGD) proteinoid polymers are produced by thermal step‐growth polymerization of R, G, and D under an inert atmosphere. P(RGD) NCs, hollow and encapsulating 25 w% each of Pal and Alp, are formed by self‐assembly of the proteinoid polymer. The encapsulation yields of Pal and Alp are 72% and 95%, respectively. Long‐term stability, controlled release, cellular uptake,synergistic cytotoxicity, and induced cell death are evident from in vitro experiments. Findings from in vivo breast, colon and gastric patient‐derived xenograft (PDX) mice experiments are consistent with the in vitro studies showing that the response to treatment with drug‐loaded NCs is similar to that elicited by free drugs, with reduced side effects. The study demonstrates the potential clinical utility of P(RGD) NCs for cancer treatment.

Supplementary Methods, Figures 1-4, Table 1 from p53 Status in Stromal Fibroblasts Modulates Tumor Growth in an SDF1-Dependent Manner

Proteinoids—simple polymers composed of amino acids—were suggested decades ago by Fox and coworkers to form spontaneously by heat. These special polymers may self-assemble in micrometer structures called proteinoid microspheres, presented as the protocells of life on earth. Interest in proteinoids increased in recent years, in particular for nano-biomedicine. They were produced by stepwise polymerization of 3–4 amino acids. Proteinoids based on the RGD motif were prepared for targeting tumors. Nanocapsules form by heating proteinoids in an aqueous solution and slowly cooling to room temperature. Proteinoid polymers and nanocapsules suit many biomedical applications owing to their non-toxicity, biocompatibility and immune safety. Drugs and/or imaging reagents for cancer diagnostic, therapeutic and theranostic applications were encapsulated by dissolving them in aqueous proteinoid solutions. Here, recent in vitro and in vivo studies are reviewed.

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine-kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second-line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib-resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

Both humoral and cellular anamnestic responses are significant for protective immunity against SARS-CoV-2. In the current study, the responses in elderly people before and after a fourth vaccine dose of BNT162b2 were compared to those of individuals immunized with three vaccine doses. Although a boost effect was observed, the high response following the third administration questions the necessity of an early fourth boost.

The efficacy/safety of combining palbociclib (a CDK4/6 inhibitor) and sunitinib (a multi-targeted receptor tyrosine kinase inhibitor) was evaluated, using patient-derived xenograft (PDX) models. Twenty-three PDX mice models were developed from patients with various solid tumors. The mice were randomized to 4 groups (5-6 mice in each): control/palbociclib (100 mg/kg)/sunitinib (50 mg/kg)/combination. Drugs were administered orally, 5 days/week. In 17/23 PDX models (74%), the combination demonstrated a synergistic inhibitory effect vs the monotherapies ("responder" models) with no unexpected toxicities. In 13/17 responder models, where standard-of-care (SOC) was an additional comparator, the combination was more effective than SOC in 7 models, as effective in 4, and less effective in 2. The mean +/- SEM experiment duration in 15/17 responder models (2/17 were excluded due to technical issues) was 86 +/- 12 and 31 +/- 5 days for the combination and control groups, respectively (p = 0.0002). The effect of the combination was dose-dependent. Cell-viability experiments in A549/MDA-MB-231/HT-29 cell lines and experiments using tumor-derived primary cell spheroids supported the PDX findings. In conclusion, combination of palbociclib and sunitinib exerts a synergistic anti-tumor effect without adding unexpected toxicity. A clinical trial assessing this combination is underway.

Objective To evaluate the durability of response 3 months after the third BNT162b2 vaccine in adults aged 60 years and older.Design Prospective cohort study.Setting Single tertiary centre.Participants Healthcare workers/family members aged ≥60 years old who received the third BNT162b2 dose.Interventions Blood samples were drawn immediately before (T0), 10–19 days (T1) and 74–103 days (T2) after the third dose.Primary and secondary outcome measures Anti-spike IgG titres were determined using a commercial assay and seropositivity was defined as ≥50 arbitrary units (AU)/mL. Neutralising antibody titres were determined at T2. Adverse events, COVID-19 infections and Clinical Frailty Scale (CFS) levels were documented.Results The analysis included 97 participants (median age, 70 years (IQR, 66–74), 58% CFS level 2). IgG titres, which increased significantly from T0 to T1 (median, 440 AU/mL (IQR, 294–923) and median, 25 429 AU/mL (IQR, 14 203–36 114), respectively; p<0.001), decreased significantly by T2, but all remained seropositive (median, 8306 AU/mL (IQR, 4595–14 701), p<0.001 vs T1). In a multivariable analysis, only time from the second vaccine was significantly associated with lower IgG levels at T2 (p=0.017). At T2, 60 patients were evaluated for neutralising antibodies; all were seropositive (median, 1294 antibody titres; IQR, 848–2072). Neutralising antibody and anti-spike IgG levels were correlated (r=0.6, p<0.001). No major adverse events or COVID-19 infections were reported.Conclusions Anti-spike IgG and neutralising antibody levels remain adequate 3 months after the third BNT162b2 vaccine in healthy adults aged ≥60 years, although the decline in IgG is concerning. A third dose of vaccine in this population should be top priority.

BACKGROUND Age and frailty are strong predictors of COVID-19 mortality. After the second BNT162b2 dose, immunity wanes faster in older ([≥]65 years) versus younger adults. The durability of response after the third vaccine is unclear. METHODS This prospective cohort study included healthcare workers/family members [≥]60 years who received a third BNT162b2 dose. Blood samples were drawn immediately before (T0), 10-19 (T1), and 74-103 (T2) days after the third dose. Antispike IgG titers were determined using a commercial assay, seropositivity was defined as [≥]50 AU/mL. Neutralizing antibody titers were determined at T2. Adverse events, COVID-19 infections, and clinical frailty scale (CFS) levels were documented. RESULTS The analysis included 97 participants (median age, 70 years [IQR, 66-74], 61% women, 58% CFS level 2). IgG titers, which increased significantly from T0 to T1 (medians, 440 AU/mL [IQR, 294-923] and 25,429 [14,203-36,114] AU/mL, respectively; P<0.001), decreased significantly by T2, but all remained seropositive (median, 8,306 AU/mL [IQR, 4595-14,701], P<0.001 vs T1). In a multivariable analysis, only time from the first vaccine was significantly associated with lower IgG levels at T2 (P=0.004). At T2, 60 patients were evaluated for neutralizing antibodies; all were seropositive (median, 1,294 antibody titer [IQR, 848-2,072]). Neutralizing antibody and antispike IgG levels were correlated (R=0.6, P<0.001). No major adverse events or COVID-19 infections were reported. CONCLUSIONS Antispike IgG and neutralizing antibodies levels remain adequate 3 months after the third BNT162b2 vaccine in healthy adults [≥]60 years, although the decline in IgG is concerning. A third vaccine dose in this population should be top priority.

AbstractBackgroundAge/frailty are strong predictors of COVID-19 mortality. After the second BNT162b2 dose, immunity wanes faster in older (≥65 years) versus younger adults. The durability of response after the third vaccine is unclear.MethodsThis prospective cohort study included healthcare workers/family members≥60 years who received a third BNT162b2 dose. Blood samples were drawn immediately before (T0), 10□19 (T1), and 74□103 (T2) days after the third dose. Anti-spike IgG titers were determined using a commercial assay, seropositivity was defined as≥50 AU/mL. Neutralising antibody titres were determined at T2. Adverse events, COVID-19 infections, and clinical frailty scale (CFS) levels were documented.FindingsThe analysis included 97 participants (median age, 70 years [IQR, 66□74], 58% CFS level 2). IgG titres, which increased significantly from T0 to T1 (medians, 440 AU/mL [IQR, 294□923] and 25,429 [14203□36114] AU/mL, respectively; p<0·001), decreased significantly by T2, but all remained seropositive (median, 8306 AU/mL [IQR, 4595□14701], p<0·001 vs T1). In a multivariable analysis, only time from the first vaccine was significantly associated with lower IgG levels at T2 (p=0·004). At T2, 60 patients were evaluated for neutralising antibodies; all were seropositive (median, 1294 antibody titre [IQR, 848□2072]). Neutralising antibody and anti-spike IgG levels were correlated (R=0·6, p<0·001). No major adverse events or COVID-19 infections were reported.InterpretationAnti-spike IgG and neutralising antibody levels remain adequate 3 months after the third BNT162b2 vaccine in healthy adults≥60 years, although the decline in IgG is concerning. A third vaccine dose in this population should be top priority.FundingNo external funding.Research in contextEvidence before this studyWe searched PubMed on Aug 1, 2021, for published research articles with no date restrictions, using the search terms of “SARS-Cov-2”, “COVID-19”, “vaccine”, “dose”, “antibody response”, and “adults” with English as a filter. Several studies were identified that investigated waning of immunity in healthy adults. It is well established through epidemiology and serology studies that in healthy adults, the protection conferred by the BNT162b2 messenger RNA (mRNA) vaccine (Pfizer/BioNtech) wanes significantly after several months. Studies have also shown that the immune response to the vaccine varies with age, and that after the second dose of the BNT162b2 vaccine, the older adult population (65-85 years of age) typically has a lower immune response (as reflected in an analysis of anti-spike IgG antibodies and neutralising antibody titres), than younger adults (18-55 years of age), and that the immunity wanes in all age groups within several months.Added value of this studyThis is, to our knowledge, the first study that examined anti-spike IgG and neutralising antibody titres three months after the third BNT162b2 vaccine dose. The study has demonstrated that three months after that dose, participants, who were healthy adults aged 60 years and older, remained anti-spike IgG seropositive, although a significant decrease in anti-spike IgG titres was observed (compared to two weeks after the third dose). In addition, a statistically significant correlation was observed between the neutralising antibody titres and the anti-spike IgG titres, and all participants were seropositive for neutralising antibodies three months after the third dose. Also, no major adverse events or COVID-19 infections were reported.Implications of all the available evidenceAs our data suggest that a third dose of the BNT162b2 vaccine is effective in maintaining adequate immune response against COVID-19 for at least several months in healthy adults aged 60 years and older, and as it is well established that older adults are at higher risk of severe COVID-19 disease and COVID-19 mortality, providing a third dose to this population should be a top priority. Our data also highlight that understanding the waning of the immune response in other age groups is key for making evidence-based policies regarding booster vaccinations for the population at large.

Personalized cancer treatment based on specific mutations offers targeted therapy and is preferred over “standard” chemotherapy. Proteinoid polymers produced by thermal step-growth polymerization of amino acids may form nanocapsules (NCs) that encapsulate drugs overcoming miscibility problems and allowing passive targeted delivery with reduced side effects. The arginine-glycine-glutamic acid (RGD) sequence is known for its preferential attraction to αvβ3 integrin, which is highly expressed on neovascular endothelial cells that support tumor growth. Here, tumor-targeted RGD-based proteinoid NCs entrapping a synergistic combination of Palbociclib (Pal) and Alpelisib (Alp) were synthesized by self-assembly to induce the reduction of tumor cell growth in different types of cancers. The diameters of the hollow and drug encapsulating poly(RGD) NCs were 34 ± 5 and 22 ± 3 nm, respectively; thereby, their drug targeted efficiency is due to both passive and active targeting. The encapsulation yield of Pal and Alp was 70 and 90%, respectively. In vitro experiments with A549, MCF7 and HCT116 human cancer cells demonstrate a synergistic effect of Pal and Alp, controlled release and dose dependence. Preliminary results in a 3D tumor spheroid model with cells derived from patient-derived xenografts of colon cancer illustrate disassembly of spheroids, indicating that the NCs have therapeutic potential.

IMPORTANCE Patients with cancer undergoing treatment are at high risk of COVID-19 following SARS-CoV-2 infection; however, their ability to produce an adequate antibody response to messenger RNA SARS-CoV-2 vaccines is unclear. OBJECTIVE To evaluate rates of antispike (anti-S) antibody response to a BNT162b2 vaccine in patients with cancer who are undergoing systemic treatment vs healthy controls. DESIGN, SETTING, AND PARTICIPANTS This prospective cohort study included 102 adult patients with solid tumors undergoing active intravenous anticancer treatment and 78 controls who received the second dose of the BNT162b2 vaccine at least 12 days before enrollment. The controls were taken from a convenience sample of the patients' family/caregivers who accompanied them to treatment. The study was conducted between February 22, 2021, and March 15, 2021 at Davidoff Cancer Center at Beilinson Hospital (Petah Tikva, Israel). INTERVENTIONS Blood samples were drawn from the study participants. Serum samples were analyzed and the titers of the IgG antibodies against SARS-CoV-2 spike receptor-binding domain were determined using a commercially available immunoassay. Seropositivity was defined as 50 or greater AU/mL. MAIN OUTCOMES AND MEASURES The primary outcomewas the rate of seropositivity. Secondary outcomes included comparisons of IgG titers and identifying factors that were associated with seropositivity using univariate/multivariable analyses. RESULTS The analysis included 180 participants, which comprised 102 patients with cancer (median [interquartile range (IQR)] age, 66 [56-72] years; 58 men [57%]) and 78 healthy controls (median [IQR] age, 62 [49-70] years; 25 men [32%]). The most common tumor type was gastrointestinal (29 [28%]). In the patient group, 92 (90%) were seropositive for SARS-CoV 2 antispike IgG antibodies after the second vaccine dose, whereas in the control group, all were seropositive. The median IgG titer in the patients with cancer was significantly lower than that in the controls (1931 [IQR, 509-4386] AU/mL vs 7160 [IQR, 3129-11 241] AU/mL; P <.001). In a multivariable analysis, the only variable that was significantly associated with lower IgG titers was treatment with chemotherapy plus immunotherapy (beta, -3.5; 95% CI, -5.6 to -1.5). CONCLUSIONS AND RELEVANCE In this cohort study of patients with cancer who were receiving active systemic therapy, 90% of patients exhibited adequate antibody response to the BNT162b2 vaccine, although their antibody titers were significantly lower than those of healthy controls. Further research into the clinical relevance of lower titers and their durability is required. Nonetheless, the data support vaccinating patients with cancer as a high priority, even during therapy.

Abstract Introduction: Treatment for cancer, particularly in later lines, is often inadequate. Combinational therapies targeting several signaling pathways simultaneously are considered a promising approach, with limited clinical evidence. Effectiveness of such combinations can be explored in patient-derived xenograft (PDX) tumor models, which more accurately represent human tumor biology, drug responsiveness, and treatment-related toxicity than standard xenograft models derived from established human cancer cell lines. Objectives: To evaluate, in PDX models, the anti-tumor effect and safety of combinational therapy including a CDK4/6 inhibitor (palbociclib) plus a multi-targeted receptor tyrosine kinase inhibitor (mtRTKI; sunitinib). Methods: Overall, 20 PDX models were developed from patients with various solid tumors. For each, tumor was implanted subcutaneously in immune-deficient mice (NRG or NSG). The mice were randomized to 4 treatment groups: vehicle as control, palbociclib (100 mg/kg), sunitinib (50 mg/kg), or combination thereof. Each treatment group included 5-6 mice (18/20 models). Drugs were administered orally, 5 days per week. Tumor volume was monitored twice a week with digital caliper. Mice body weight and clinical signs were examined twice a week to evaluate toxicity. Results: The mean experiment time (all experiments) was 66 days (SEM, 10 days; range, 15-170 days). In 14 of the 20 evaluated models (70%), including those for cholangiocarcinoma, breast, lung, pancreas, colon, stomach, and ovarian cancer, combinational therapy with palbociclib and sunitinib had significantly superior anti-cancer activity compared to either drug alone, without any unexpected toxicity. Mean duration of response in these 14 models was 72 days (SEM, 12 days). The combination of palbociclib and sunitinib prevented tumor growth, and in 3 models reduced tumor size. In the 2 PDX models developed from patients with palbociclib-resistant disease, the combination overcame tumor resistance to palbociclib. In 5 of the models which demonstrated synergy between palbociclib and sunitinib, the effect of administrating 2-fold lower doses of both drugs was evaluated. The lower doses were shown to suppress tumor growth in a dose-response manner. Conclusions: These PDX results suggest that the combination of palbociclib and sunitinib exert a synergistic anti-tumor effect on multiple tumor types without adding unexpected toxicity. Citation Format: Neta Moskovits, Idit Peretz, Eva Chausky, Raisa Meerson, Ella Itzhaki, Noga Marsiano, Nofar Shmuel, Ranny Yaffe, Natalia Edison, Tal Goldman, Salomon Stemmer. Synergistic anti-cancer activity of palbociclib in combination with sunitinib in patient-derived xenograft (PDX) models of various human cancers types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5054.

Personalized cancer immunotherapy targeting patient-specific cancer/testis antigens (CTA) and neoantigens may benefit from large-scale tumor human leukocyte antigen (HLA) peptidome (immunopeptidome) analysis, which aims to accurately identify antigens presented by tumor cells. Although significant efforts have been invested in analyzing the HLA peptidomes of fresh tumors, it is often impossible to obtain sufficient volumes of tumor tissues for comprehensive HLA peptidome characterization. This work attempted to overcome some of these obstacles by using patient-derived xenograft tumors (PDX) in mice as the tissue sources for HLA peptidome analysis. PDX tumors provide a proxy for the expansion of the patient tumor by re-grafting them through several passages to immune-compromised mice. The HLA peptidomes of human biopsies were compared with those derived from PDX tumors. Larger HLA peptidomes were obtained from the significantly larger PDX tumors as compared with the patient biopsies. The HLA peptidomes of different PDX tumors derived from the same source tumor biopsy were very reproducible, even following subsequent passages to new naïve mice. Many CTA-derived HLA peptides were discovered, as well as several potential neoantigens/variant sequences. Taken together, the use of PDX tumors for HLA peptidome analysis serves as a highly expandable and stable source of reproducible and authentic peptidomes, opening up new opportunities for defining large HLA peptidomes when only small tumor biopsies are available. This approach provides a large source for tumor antigens identification, potentially useful for personalized immunotherapy.

Extracellular matrix (ECM) plays an important role in tumor development and dissemination, but few points of therapeutic intervention targeting ECM of the tumor microenvironment have been exploited to date. Recent observations suggest that the enzymatic introduction of disulfide bond cross-links into the ECM may be modulated to affect cancer progression. Specifically, the disulfide bond-forming activity of the enzyme Quiescin sulfhydryl oxidase 1 (QSOX1) is required by fibroblasts to assemble ECM components for adhesion and migration of cancer cells. Based on this finding and the increased QSOX1 expression in the stroma of aggressive breast carcinomas, we developed monoclonal antibody inhibitors with the aim of preventing QSOX1 from participating in pro-metastatic ECM remodeling. Here we show that QSOX1 inhibitory antibodies decreased tumor growth and metastasis in murine cancer models and had added benefits when provided together with chemotherapy. Mechanistically, the inhibitors dampened stromal participation in tumor development, as the tumors of treated animals showed fewer myofibroblasts and poorer ECM organization. Thus, our findings demonstrate that specifically targeting excess stromal QSOX1 secreted in response to tumor-cell signaling provides a means to modulate the tumor microenvironment and may complement other therapeutic approaches in cancer.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death worldwide, with poor outcome of current treatments. Major contributors to therapeutic resistance in PDAC include Kras mutations, a dense desmoplastic stroma, and activation of feedback signaling pathways. STAT3 and Insulin Receptor Substrate 1/2 (IRS1/2) regulate tumor survival and metastasis, the crosstalk of the tumor and its microenvironment, and play major role in drug resistance. NT219 is a novel bispecific inhibitor of IRS1/2 and STAT3, leading to IRS1/2 elimination and STAT3 dephosphorylation. The purpose of this study was to assess NT219 activity in PDAC PDX models and define schedule and regimens and combinations to overcome resistance to optimize future clinical protocols. Methods: Patient-derived tumor xenograft (PDX) models of mutated KRAS pancreatic cancer were used to test the efficacy of NT219 in combination with gemcitabine, trametinib, and folfirinox. RNAseq was used to analyze the pharmacodynamic effect of NT219. Pharmacokinetic profile of NT219 was tested. Results: Chemoresistent tumors of 4 PDAC PDX models were exposed to NT219 with gemcitabine demonstrated reversal of pre-existing resistance. One of these models demonstrated complete response in 5 out of 10 mice upon addition of NT219 to gemcitabine. Similar results were observed when NT219 was combined with trametinib (a MEK inhibitor) and with folfirinox (chemotherapy). There was a correlative dose-escalation response of both plasma and tumor levels and the therapeutic effect. NT219 delivered prior to gemcitabine showed a much better response than the inverse schedule/drug administration. RNAseq analysis of the tumors revealed that the combined treatment with NT219 and gemcitabine lowered the levels of IRS1 to 20% of the control group. Similar reductions were observed for STAT3-regulated genes, as well as for Ki67 (a proliferation marker), cyclin D (prognostic marker), and TGFβ (driver of epithelial to mesenchymal transition). Conclusion: NT219 overcomes gemcitabine acquired resistance in PDAC. The combination of NT219 with modern chemotherapy in pancreatic cancer may enhance efficacy and delay acquired resistance in this fatal disease. Citation Format: Hadas Reuveni, Lana Kupershmidt, Neta Moskovits, Evgeny Solomonov, Salomon M Stemmer, Izhak Haviv. NT219, a novel bispecific inhibitor of STAT3 and IRS1/2, combined with chemotherapy or MEK inhibitor in gemcitabine-resistant pancreatic tumors, induced tumor regression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C45.

Abstract Background: Circulating tumor cells (CTCs) originate from both primary and metastatic solid tumors and infiltrate the blood system. Although the metastatic potential of the single CTC cell is unknown, CTCs in circulation have been reported to have prognostic value as early predictors of relapse in cancers including ovarian, breast, colorectal, prostate, lung, bladder, head and neck and melanoma. As metastatic spread remains the major problem in cancer treatment, it is of great importance to characterize the subpopulations of CTCs with the highest metastatic potential. Methods: Peripheral blood mononuclear cells (MNCs) or bone marrow MNCs, were collected from each of over 60 cancer patients and grafted separately to immunodeficient NOD.Cg-Prkdc.scid.Il2rgtm1Wjl/SzJ (NSG) mice. Additionally, CTCs were enriched by either CD8 T-cell depletion or by a size-based filtration system. Tumors that developed in the mice were confirmed to be human tumors by demonstrating that they have human HLA expression and by comparing their tumor type by an independent pathologist to that of the donor patient's. The CTC-derived tumors were passaged to naive NSG mice to establish the scale up potential for treatment-groups. To facilitate future research of CTC-derived tumors, we have established a Biobank for CTC-derived tumors with matched samples of the patient's MNCs, plasma, tumor biopsy(s) and patient-biopsy derived xenograft (PDX) tumors. Additional characterization of the CTC-derived tumors was performed, such as comparison to the driver-mutations of the original tumor-biopsies and expression of epithelial or mesenchymal markers. The clinical relevance of the CTC-derived tumors was assessed by long term follow-up of each patient's disease progression. Results: CTC-derived tumors were detected in xenografts in 15 cases, within an average of eight months post grafting, representing a 25% success rate. The CTC-derived tumors were from a variety of cancers including: breast (6), colon (1), lung (2), pancreatic (1), ovarian (1), malignant-mesothelioma (1) head and neck (1) hepatocellular carcinoma (1) and sarcoma (1). The patients, whose blood or marrow contained tumorigenic CTCs, developed metastasis. Interestingly, there was a correlation between sites of CTC-derived tumors in the mice and sites of metastatic spread in the patients. CTC-derived tumors were successfully transferred to subsequent PDX mice, enabling the establishment of large treatment groups to study drug reactivity of these tumors. Summary: This study describes a functional assay to characterize the CTCs subtypes with a high metastatic potential, which utilizes the capacity of CTCs to form tumors in a permissive immune-free environment. CTC-derived tumors may offer a new tool to predict the significance of these malignant cells and prevent metastatic spread. Citation Format: Netta R. Shraga, Amihai Lieberman, Neta Moskovits, Alejandro Livoff, Evgeny Solomonov, Inbar Ben Shachar, Aron Popovtzer, Salomon M. Stemmer*, Izhak Haviv*. Circulating tumor cells from patients' blood induce tumors in a personalized xenograft mouse model: A functional approach to assess the patients' premetastatic disease state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1030.

Abstract Feedback activation of STAT3 and IGF1R/IRS plays a prominent role in mediating drug resistance to a broad spectrum of targeted cancer therapies and chemotherapies. Both the IRS1/2 and STAT3 are major signaling junctions regulated by various oncogenes, and altered during EMT and drug resistance. STAT3 has also been known to play an active role in immune-evasion of tumors and inhibition of STAT3, both in the tumor, as well as in the tumor's microenvironment, may therefore potentiate immune attack on the tumor. NT-219 is a dual inhibitor of STAT3 and IRS1/2 developed by TyrNovo Ltd. to overcome cancer drug resistance. NT-219 inhibits STAT3 phosphorylation and eliminates IRS1/2 in a unique 3-step mechanism: dissociation of IRS1/2 from the IGF1 receptor, induction of IRS1/2 serine phosphorylation, and subsequent degradation by the proteasome. We recently demonstrated that the inhibition of both IRS and STAT3 are required and essential for overcoming drug resistance. NT-219 efficacy was demonstrated in Patient-Derived tumor Xenograft (PDX) models of multiple cancer types: melanoma, sarcoma, pancreatic, colon, lung, and head & neck, when added-on to the approved therapies. In these models, NT-219 overcame acquired resistance to several oncology drug families: inhibitors of EGFR (Tarceva®, Erbitux®, Tagrisso®), MEK (Mekinist®), mutated-BRAF (Zelboraf®), mTOR (Afinitor®) as well as with chemotherapy agents (Gemzar®, 5FU, Oxaliplatin). We recently demonstrated that NT-219 works in synergy also with immune-oncology therapies. By using double autologous PDX models we demonstrated that NT-219 converted non-responding tumors to responders to Keytruda®. It also enhanced the immunotherapeutic potential of Cetuximab. The unique mode of action of NT219 may open a new avenue of combined targeted therapies in a wide range of malignancies, and has the potential to expand response duration and target patient population to the applicable drugs. Citation Format: Lana Kuperschmidt, Hadas Reuveni, Shani Carmi, Neta Moskovits, Netta R. Shraga, Evgeny Solomonov, Ohad Ronen, Salomon Stemmer, Izhak Haviv. NT219, a novel dual inhibitor of STAT3 and IRS1/2, converts immuno-oncology resistant tumors to responders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2754.

Abstract The p53 tumor suppressor exerts a variety of cell-autonomous effects that are aimed to thwart tumor development. In addition, however, there is growing evidence for cell nonautonomous tumor suppressor effects of p53. In the present study, we investigated the impact of stromal p53 on tumor growth. Specifically, we found that ablation of p53 in fibroblasts enabled them to promote more efficiently the growth of tumors initiated by PC3 prostate cancer-derived cells. This stimulatory effect was dependent on the increased expression of the chemokine SDF-1 in the p53-deficient fibroblasts. Notably, fibroblasts harboring mutant p53 protein were more effective than p53-null fibroblasts in promoting tumor growth. The presence of either p53-null or p53-mutant fibroblasts led also to a markedly elevated rate of metastatic spread of the PC3 tumors. These findings implicate p53 in a cell nonautonomous tumor suppressor role within stromal fibroblasts, through suppressing the production of tumor stimulatory factors by these cells. Moreover, expression of mutant p53 by tumor stroma fibroblasts might exert a gain of function effect, further accelerating tumor development. Cancer Res; 70(23); 9650–8. ©2010 AACR.