Tumor heterogeneity drives drug resistance and relapse, influencing immune evasion and tumor progression. While intratumor heterogeneity has been extensively studied at the genomic level, its functional outcomes and interactions with the tumor microenvironment remain underexplored. In contrast, the functional outcome of heterogeneity and the interplay with the tumor microenvironment have not been addressed. In this study, we integrate multi-region spatial MS-based proteomics of 280 tumor regions, exome sequencing, and imaging to investigate spatial proteomic heterogeneity in breast cancer. Our findings reveal increased proteomic heterogeneity with tumor progression, independent of genomic heterogeneity but closely associated with microenvironmental differences. Integration with immune and stromal imaging highlighted a dynamic interplay where low-grade tumors exhibit constrained immune infiltration, and upon progression to higher grades, macrophages and T cells infiltrate. However, anti-inflammatory pathways involving kynurenine and prostaglandins are more highly expressed in infiltrated regions, suggesting that anti-tumorigenic activities are inhibited. Integration with the global protein network provides potential targetable mediators of immune evasion in breast cancer that can serve as the basis for future development of personalized breast cancer therapies.

This study explores the utilization of digital light processing (DLP) printing to fabricate complex structures using native gelatin as the sole structural component for applications in biological implants. Unlike approaches relying on synthetic materials or chemically modified biopolymers, this research harnesses the inherent properties of gelatin to create biocompatible structures. The printing process is based on a crosslinking mechanism using a di-tyrosine formation initiated by visible light irradiation. Formulations containing gelatin were found to be printable at the maximum documented concentration of 30 wt.%, thus allowing the fabrication of overhanging objects and open embedded tubes with a compressive modulus akin to soft tissues. Cell adhesion and growth onto and within the gelatin-based 3D constructs were evaluated by examining two implant fabrication techniques: (1) cell seeding onto the printed scaffold and (2) printing compositions that contain cells (cell-laden). The preliminary biological experiments indicate that both the cell-seeding and cell-laden strategies enable making 3D cultures of chondrocytes within the gelatin constructs. This study underscores the potential of utilizing non-modified protein-only bioinks in DLP printing to produce intricate 3D objects with high fidelity, paving the way for advancements in regenerative tissue engineering.

This study explores the utilization of digital light processing (DLP) printing to fabricate complex structures using native gelatin as the sole structural component for applications in biological implants. Unlike approaches relying on synthetic materials or chemically modified biopolymers, this research harnesses the inherent properties of gelatin to create biocompatible structures. The printing process is based on a crosslinking mechanism using a di-tyrosine formation initiated by visible light irradiation. Formulations containing gelatin were found to be printable at the maximum documented concentration of 30 wt.%, thus allowing the fabrication of overhanging objects and open embedded. Cell adhesion and growth onto and within the gelatin-based 3D constructs were evaluated by examining two implant fabrication techniques: (1) cell seeding onto the printed scaffold and (2) printing compositions that contain cells (cell-laden). The preliminary biological experiments indicate that both the cell-seeding and cell-laden strategies enable making 3D cultures of chondrocytes within the gelatin constructs. The mechanical properties of the gelatin scaffolds have a compressive modulus akin to soft tissues, thus enabling the growth and proliferation of cells, and later degrade as the cells differentiate and form a grown cartilage. This study underscores the potential of utilizing non-modified protein-only bioinks in DLP printing to produce intricate 3D objects with high fidelity, paving the way for advancements in regenerative tissue engineering.

PDF file - 43K, the objective responses of individual patients to various types of immunotherapies.

PDF file - 74K, the baseline and treatment characteristics of evaluated TIL patients treated with or without ipilimumab before TIL ACT.

PDF file - 60K, the baseline characteristics and response to ipilimumab in 19 patients previously treated with TIL ACT.

CCR Translation for This Article from Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma: Intent-to-Treat Analysis and Efficacy after Failure to Prior Immunotherapies

PDF file - 112K, the correlation of different variables to one another and their p values.

Supplementary Data from Clinical Responses in a Phase II Study Using Adoptive Transfer of Short-term Cultured Tumor Infiltration Lymphocytes in Metastatic Melanoma Patients

SummaryDespite extensive research, internal tumor heterogeneity presents enormous challenges to achieve complete therapeutic responses. Changes in protein expression are central determinants of cancer phenotypes that reflect potential therapeutic targets. However, previous proteomic studies did not address internal heterogeneity, therefore, masked the necessary spatial resolution to achieve a comprehensive understanding of cancer complexity. Here we present the first large-scale multi-focal breast cancer proteomic study of 330 tumor regions which associated cancer cell function, pathological parameters, and spatial localization of each tumor region. We found marked internal proteomic heterogeneity even within tumors presenting homogeneous receptor expression. Additionally, analysis of the internal heterogeneity, based on coexisting receptor expression or histological patterns in single tumors, showed significant functional differences between homogeneous and heterogeneous tumors related to cancer metabolism, immunogenicity, and proliferation. We anticipate that this study will serve as a starting point towards the development of improved cancer therapy and diagnostics.

We aimed to immunohistochemically characterize the pattern of expression of epithelial markers in rare head and neck squamous cell carcinoma (HNSCC) variants: carcinoma cuniculatum (CC) and adenosquamous carcinoma (ASC). We also present an additional variant of HNSCC with concomitant basaloid and squamous components that has overlapping morphological features with odontogenic and non-odontogenic tumors, which we termed basalo-squamous carcinoma (BSC). The selected markers included CK5/6, p40, CK19, BerEP4, p16 and SOX10. All tumors were CK5/6 and p40 positive. CK19 and BerEP4 were positive in BSC and focally in ASC but negative in CC. p16 was positive in 3 (60%) of the CCs, focally positive in ASC and negative in BSC. SOX10 was negative in all three variants. Our results highlight the plasticity of the lining epithelium revealing differential profiles of immuno-expression of the selected molecular markers, possibly reflecting their diverse histopathogenesis.

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.

The location of Warthin tumor (WT) in the parotid gland impacts the surgical approach and may be indicative of the elusive origin of this intriguing entity. Location in the deep versus superficial lobe of the gland is not directly addressed when defining WT characteristics. Our observation, of rare occurrence of deep lobe WT, if at all, led to the current investigation. The study design is cohort study. This is a retrospective chart review of all patients undergoing parotidectomy for WT in two tertiary academic referral centers: the Sheba Medical Center (SMC), Israel, and the Christiana Care (CC), Newark, Delaware, USA. 122 consecutive adult patients underwent parotidectomy for WT (72 from SMC and 50 from CC). Seventy percent were males, with a mean age of 60.6 years. Bilateral WT or multi-centric WT were found in 9.8 and 17.2% of the cases, respectively. In one case, the tumor was described as originating in the deep lobe. In all other cases, the tumor originated and was limited to the superficial lobe. 99.2% of WT originated in the superficial lobe, corresponding with the few reports directly addressing its location in the gland. The reason for the tumor to be limited almost uniformly to the superficial lobe is unknown, and could be related to the etiopathogenesis of this elusive entity. We suggest adding tumor location within the superficial lobe to the common characteristics of WT (male, smoking, and lower pole) that serve as “common criterion” while evaluating a parotid lesion.

ObjectiveCorrect interpretation of incidental tumors is important to plan an appropriate treatment. We assessed the incidence and imaging characteristics of fluorine-18 fluorodeoxyglucose (F-18-FDG)-avid focal parotid findings (FPFs) in patients with lung cancer.Patients and methodsFPFs in PET-computed tomography reports of cancer patients were searched. Those with known parotid malignancies, lymphoma, and diffuse F-18-FDG uptake in the entire parotid gland were not included in the analysis.ResultsFPFs were detected in 38/3120 cancer patients (1.23%), observed as a soft tissue mass with a mean diameter 1.60.5cm (range 0.8-2.7cm) and a mean maximum standardized uptake value of 7.7 +/- 3.7 (range 2.5-17.8). FPFs were observed in 23/604 (3.8%) patients with lung cancer, compared with 6/1366 (0.4%) with breast cancer and 5/842 (0.6%) with gastrointestinal malignancies. We assessed FPFs appearances in 23 patients with lung cancer (18 men, mean age 72.8 +/- 9.2); 20 (87%) were current or past smokers. There was no correlation between the stage or histopathological type of the lung cancer and the prevalence of parotid lesions. In four patients with histopathology, no malignancy was detected. For an additional 11 patients with available imaging and clinical follow-up (mean follow-up 15.5 +/- 13.5 months, range 3-42 months), FPFs were consistent with benign lesions.ConclusionFPFs were more prevalent among patients with lung cancer than in patients with other malignancies. As F-18-FDG avidity was moderate to high, FPFs may mimic distant metastases. It is important to consider FPFs in the interpretation of a focal parotid lesion as misinterpretation may result in denial of appropriate therapy.

Abstract Purpose: Adoptive cell transfer (ACT) using autologous tumor-infiltrating lymphocytes (TIL) was reported to yield objective responses in about 50% of metastatic patients with melanoma. Here, we present the intent-to-treat analysis of TIL ACT and analyze parameters predictive to response as well as the impact of other immunotherapies. Experimental Design: Eighty patients with stage IV melanoma were enrolled, of which 57 were treated with unselected/young TIL and high-dose interleukin-2 (IL-2) following nonmyeloablative lymphodepleting conditioning. Results: TIL cultures were established from 72 of 80 enrolled patients. Altogether 23 patients were withdrawn from the study mainly due to clinical deterioration during TIL preparation. The overall response rate and median survival was 29% and 9.8 months for enrolled patients and 40% and 15.2 months for treated patients. Five patients achieved complete and 18 partial remission. All complete responders are on unmaintained remission after a median follow-up of 28 months and the 3-year survival of responding patients was 78%. Multivariate analysis revealed blood lactate-dehydrogenase levels, gender, days of TIL in culture, and the total number of infused CD8+ cells as independent predictive markers for clinical outcome. Thirty-two patients received the CTLA-4-blocking antibody ipilimumab prior or post TIL infusion. Retrospective analysis revealed that nonresponders to ipilimumab or IL-2 based therapy had the same overall response rate to ACT as other patients receiving TIL. No additional toxicities to TIL therapy occurred following ipilimumab treatment. Conclusion: Adoptive transfer of TIL can yield durable and complete responses in patients with refractory melanoma, even when other immunotherapies have failed. Clin Cancer Res; 19(17); 4792–800. ©2013 AACR.

Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts poor prognosis in melanoma patients. Inhibition of this alternative vascularization pathway might be of clinical importance, especially as several anti-angiogenic therapies targeting endothelial cells are largely ineffective in melanoma. We show the presence of VM structures histologically in a series of human melanoma lesions and demonstrate that cell cultures derived from these lesions form tubes in 3D cultures ex vivo. We tested the ability of nicotinamide, the amide form of vitamin B3 (niacin), which acts as an epigenetic gene regulator through unique cellular pathways, to modify VM. Nicotinamide effectively inhibited the formation of VM structures and destroyed already formed ones, in a dose-dependent manner. Remarkably, VM formation capacity remained suppressed even one month after the complete withdrawal of Nicotimamid. The inhibitory effect of nicotinamide on VM formation could be at least partially explained by a nicotinamide-driven downregulation of vascular endothelial cadherin (VE-Cadherin), which is known to have a central role in VM. Further major changes in the expression profile of hundreds of genes, most of them clustered in biologically-relevant clusters, were observed. In addition, nicotinamide significantly inhibited melanoma cell proliferation, but had an opposite effect on their invasion capacity. Cell cycle analysis indicated moderate changes in apoptotic indices. Therefore, nicotinamide could be further used to unravel new biological mechanisms that drive VM and tumor progression. Targeting VM, especially in combination with anti-angiogenic strategies, is expected to be synergistic and might yield substantial anti neoplastic effects in a variety of malignancies.

Treatment of metastatic melanoma patients with adoptively transferred tumor infiltrating lymphocytes (TIL) has developed into an effective therapy. Various studies reported objective responses of 50% and more. The use of unselected, minimally cultured, bulk TIL (Young-TIL) has simplified the TIL production process and may therefore, allow the accessibility of this approach to cancer centers worldwide. This article describes the precise process leading to the large-scale production of Young-TIL for therapy. We have enrolled 55 melanoma patients and optimized their Young-TIL generation process. Young-TIL cultures were successfully established for 51 of 55 (93%) patients in 16.7±5.5 days. In a large-scale expansion procedure Young-TIL of 32 patients were further expanded to treatment levels, resulting in a final number of 4.5×1010 ±2.0×1010 TIL. Fifteen of 31 (48%) patients, who were evaluated, achieved a clinical response, including 4 complete and 11 partial responses. We confirmed the significant correlation between short culture duration, high number of infused cells, and tumor regression. A high percentage of CD8+ T cells in the infusion product was beneficial to achieve an objective response. All responding patients were treated with Young-TIL cultures established in <20 days. In summary, we describe here an efficient and reliable method to generate Young-TIL for adoptive transfer therapy, which may easily be adopted by other cancer centers and can lead to objective responses in 50% of refractory melanoma patients. In the future this approach may be used also in other types of malignancies.