Highly multiplexed 3D profiling of cell states and immune niches in human tumors

Diseases like cancer involve alterations in in cell proportions, states, and local interactions as well as complex changes in 3D tissue architecture. However, disease diagnosis and most multiplexed spatial profiling studies rely on inspecting thin (4–5 micron) tissue specimens. Here, we use confocal microscopy and cyclic immunofluorescence (3D CyCIF) to show that few if any cells are intact in these thin sections; this reduces the accuracy of cell phenotyping and interaction analysis. In contrast, high-plex 3D CyCIF imaging of intact cells in thick tissue sections enables accurate quantification of marker proteins and detailed analysis of intracellular structures and organelles. Precise imaging of cell membranes also makes it possible to detect juxtacrine signalling among interacting tumour and immune cells and reveals the formation of spatially-restricted cytokine niches. Thus, 3D CyCIF provides insights into cell states and morphologies in preserved human tissues at a level of detail previously limited to cultured cells.

ABSTRACTRegulation of cell architecture is critical in the formation of tissues during animal development. The mechanisms that control cell shape must be both dynamic and stable in order to establish and maintain the correct cellular organization. Previous work has identified Shroom family proteins as essential regulators of cell morphology during vertebrate development. Shroom proteins regulate cell architecture by directing the subcellular distribution and activation of Rho-kinase, which results in the localized activation of non-muscle myosin II. Because the Shroom-Rock-myosin II module is conserved in most animal model systems, we have utilized Drosophila melanogaster to further investigate the pathways and components that are required for Shroom to define cell shape and tissue architecture. Using a phenotype-based heterozygous F1 genetic screen for modifiers of Shroom activity, we identified several cytoskeletal and signaling protein that may cooperate with Shroom. We show that two of these proteins, Enabled and Short stop, are required for ShroomA-induced changes in tissue morphology and are apically enriched in response to Shroom expression. While the recruitment of Ena is necessary, it is not sufficient to redefine cell morphology. Additionally, this requirement for Ena appears to be context dependent, as a variant of Shroom that is apically localized, binds to Rock, but lacks the Ena binding site, is still capable of inducing changes in tissue architecture. These data point to important cellular pathways that may regulate contractility or facilitate Shroom-mediated changes in cell and tissue morphology.

Mechanical properties of the lungs characterize the restoring forces of the passively distended lung. Inside a recently described bioreactor we applied cyclic strain on lung tissue (dynamic mechanostimulation). Deflection of the tissue was alternated in a sinusoidal time course by application of gas volume into the pressure chamber of the bioreactor, using a ventilator for small animals. Self-made air-tight highly pliant siloxane-membranes served as carrier membranes for displacement of the tissue inside the bioreactor. Pressure-volume loops resulting from the counterforce of the tissue probes were measured and analyzed. Using a newly developed technique for assessment of dynamic microscopy images we demonstrated changes in tissue morphology during the dynamic conditions of cyclic strain.The bioreactor was fixed centrally under a microscope. The vertical deflection range of the tissue was subdivided into 7 steps of microscope focusing. For each of the 7 microscope focus positions a movie sequence was recorded during deflection cycles. From each movie the respective image with best image definition was extracted. The resulting images were sorted and synchronized to their respective temporal position inside the deflection cycle.An average size of tissue units was estimated for each image. Pressure-volume curves of the tissue were calculated after subtracting that fraction of pressure that was caused by the carrier membrane.Nonlinear mechanical characteristics of the tissue probes could be demonstrated during dynamic conditions. Corresponding changes of local morphology of the tissue were documented.We conclude that our new method for Dynamic Videomicroscopy allows to evaluate changes in tissue morphology during dynamic mechanostimulation. Comparison to tissue morphology during static mechanostimulation could improve the understanding of macroscopic mechanical characteristics of soft biological tissues.Keywordsmechanostimulationrespiratory systemalveolar structurepressure-volume relationship

To observe the phenomenon of apoptosis and expression of related proteins in injured muscle tissue during the formation of pressure injury, and to explore its mechanism of action on the pressure injury. Forty male Sprague-Dawley (SD) rats were divided into normal control group, 3, 5, 7 and 9 compression groups according to the random number table, with 8 rats in each group. The pressure injury models on the gracilis muscle of hind limbs were reproduced by using a way of cycle compression of ischemia/reperfusion (I/R) magnet. One cycle consisted of 12-hour compression, and followed by 12-hour release. The 5, 7, 9 compression groups were cut through after receiving 3 cycles. The normal control group did not receive any treatment. Muscle tissue specimens were harvested in the pressurized center at the end of the experiment (the same site for the normal control group), then the rats were sacrificed. The hematoxylin-eosin (HE) staining was used to examine the changes of muscle tissue morphology. The Hoechst 33258 staining was used to evaluate the apoptosis of muscle tissue. Western Blot was used to detect the expressions of heat shock protein 70 (HSP70), B-cell lymphoma-2 protein (Bcl-2) and Bcl-2 associated X protein (Bax). (1) HE staining showed that the compressed tissues appeared different degrees of pathological degradation, and the change of tissue morphology was more serious with the increase of the compression cycle. (2) Hoechst 33258 staining showed that the nuclei of compressed tissue showed condensed, compact morphology and granular fluorescence, and the number of apoptotic cells increased with the increase of the compression cycle. (3) Western Blot showed that with the increase of the compression cycle the protein expressions of HSP70 and Bax were gradually increased, and the protein expression of Bcl-2 was gradually decreased. Compared with the normal control group, the protein expression of HSP70 in the 9 compression group was increased with statistically significant differences (HSP70/GAPDH: 1.78±0.21 vs. 0.55±0.17, P < 0.01). The protein expression of Bax in the 7 and 9 compression groups were increased with statistically significant differences (Bax/GAPDH: 0.96±0.09, 0.98±0.02 vs. 0.67±0.07, both P < 0.01). The protein expression of Bcl-2 in the 3, 5, 7 and 9 compression groups were significantly decreased (Bcl-2/GAPDH: 0.17±0.03, 0.13±0.03, 0.14±0.03, 0.10±0.02 vs. 0.36±0.04, all P < 0.05). Apoptosis can be induced by I/R in pressure injury tissues. Apoptosis induced by HSP70 and apoptosis factors Bcl-2 and Bax may be involved in the formation of pressure injury.

The effects of food processing and or cooking practices in the variations in morphological changes in tissues were investigated. The relationship between consumption of charred meat, which is believed to be rich in nitrosamine, by pregnant mothers and the adverse effects on the growth of their offsprings, alterations in morphology of tissues like liver and pancreas were studied. Meat was subjected to charcoal fire roasting without curing and was thereafter fed to pregnant rats. The results showed growth retardation of the offsprings, micromorphological changes in tissues such as liver [generalized apoptotic processes and hepatocellular necrosis] and pancreas [increased islet cells density and scattered acinar hyperplasia with solid cellular area] in the offsprings of the female albino rats that were fed on 60 and 80 percent of charred meat regimen during gestation and lactation periods when compared to control. These observations have shown that meat cured or uncured when subjected to charcoal fire roasting may cause alteration in the morphology of the foetal tissues.

Evidence for a plasma membrane redox system on intact ascites tumor cells with different metastatic capacity

Multiplexing in immunofluorescence imaging is important for the spatial profiling of cells and molecules in tumor tissue samples. Cyclic immunofluorescence (IF) methods using oxidants (e.g. hydrogen peroxide) and enzymes (e.g. DNase) localize a great number of cellular makers and proteins in a tissue section while repeating a process of IF staining, imaging, and fluorescence deactivation. However, the repeated use of chemicals and enzymes might cause artifacts in tissue and cell morphologies. Furthermore, these methods are restricted to thin tissue sections (~5 μm thick) which are inappropriate to provide comprehensive structural information on tissue samples. Although reconstruction of two-dimensional (2D) images from serial tissue sections can provide a certain volumetric tissue image, it takes a huge amount of time and effort. Here we introduce a three-dimensional (3D) multiplex IF imaging method using LED photobleaching. We built high-power LED illuminators with 100W warm (emission wavelength: 480-700 nm), green (430-520 nm), and red (600- 680 nm) LED chips, which can efficiently bleach a broad or selected wavelength of fluorescence signals in tissue samples. We integrated this LED photobleaching with the Transparent Tissue Tomography (T3) protocol and created a 3D cyclic IF method involving tissue macrosectioning (400 μm), three-color IF staining, D-fructose-based tissue clearing, 3D confocal fluorescence microscopy, LED photobleaching, tissue washing, and three-color IF staining for other biomarkers, and repeating the process. By applying this method to mouse mammary tumor tissues, we could perform 8-plex fluorescence microscopy for visualizing cell nuclei (DAPI), vascular (CD31, SMA) and structural (ER-TR7) cells, immune cells (CD3, CD8, CD45), and cancer cells (CK8) in the tumor macrosections in 3D at tissue and cellular resolution. To validate the method as an evaluation tool for immunotherapy, we treated the mouse mammary tumor with a STING agonist (DMAXX) intratumorally and collected the tumor tissue 1 day after the treatment, and processed it for the 3D cyclic IF protocol. The quantitative multiplex image data showed immune-driven-cancer eradication and high tumor infiltration of a large number of CD3+CD8+CD45+ cytotoxic T cells. We also examined that Red and Green LED illumination can selectively bleach fluorophores in tissues, which would be useful for patterning fluorescence in tissue as well as studying fluorescent drug-cell interaction in a tissue. In summary, this chemical and enzyme-free 3D cyclic IF imaging method will be a powerful tissue assay tool to provide comprehensive spatial information of tissue (tumor) samples including cell types, cellular and molecular location, and their 3D organization in a tissue sample. Citation Format: Jingtian Zheng, Evan Phillips, Yi-Chien Wu, Steve Seung-Young Lee, Vytautas Bindokas. LED photobleaching-based multiplex 3D microscopy of the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4707.

Kinetics of amidophosphoribosyltransferase in intact tumor cells

Across developmental systems, quantitative and imaging-based approaches have provided unprecedented resolution of dynamic changes in gene regulation and cell fate specification, along with complex changes in tissue morphology. This has set the stage for a wealth of comprehensive theoretical models, parameterised by experimental data, able to reproduce key aspects of biological behaviour and jointly enabling a higher level of abstraction, going from the identification of the molecular components to understanding complex functional relationships between these components. Despite these successes, gaining a cross-scale understanding of developmental systems will require further collaboration between disciplines, from developmental biology to bioengineering, systems biology and biophysics. We highlight the exciting multi-disciplinary research discussed at The Company of Biologists workshop 'Fostering quantitative modelling and experimentation in Developmental Biology'.

&lt;div&gt;Abstract&lt;p&gt;Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies.&lt;/p&gt;Significance:&lt;p&gt;The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies.&lt;/p&gt;Significance:&lt;p&gt;The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.&lt;/p&gt;&lt;/div&gt;

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.

Regulation of enzymes of purine metabolism in intact tumor cells

The goal of our research is to develop an approach to early detection and diagnosis of oral neoplasia using opticalbased technologies such as fluorescence spectroscopy and imaging, high-resolution microendoscopy and molecularly-targeted optical contrast agents. Although patients with early disease have better chances for cure and functional outcome, most patients present with advanced tumours when treatment is less successful and often causes severe deficits in speech, swallowing, facial appearance and quality of life. To improve outcomes we must improve detection and diagnosis of early neoplastic changes. Changes in tissue architecture, morphology, and molecular composition that occur during carcinogenesis also produce changes in the optical properties of tissue. These changes can be detected non-invasively, in vivo, and in near real-time using optical spectroscopy and imaging. Our research group has developed multi-spectral imaging devices for wide field visualization of early changes in oral mucosa. We are investigating whether these devices can improve the clinician's ability 1) to improve visualization of early neoplastic changes in oral mucosa that can be difficult to see using normal white light examination in community settings, 2) to choose optimal sites within lesions to perform biopsies, and 3) to visualize the peripheral margins of disease. We have also shown that using computer algorithms and disease probability maps, optical imaging and spectroscopy can provide objective discrimination between normal and abnormal oral mucosa with good sensitivity and specificity – similar to that of oral cancer specialists. We are now developing simplified optical devices to use in screening and diagnosis in community settings.

10596 Background: The vascular endothelial growth factors (VEGFs) -A, -C, -D and the vascular endothelial growth factor Receptors (VEGFRs) -1, -2 and -3 are important molecular markers in angiogenesis and lymphangiogenesis. This study elucidates the prognostic significance of these molecular markers in tumor cells as well as in the tumor stroma of resected NSCLC tumors. Methods: Tumor tissue samples from 335 resected patients with stage I to IIIA were obtained and tissue microarrays were constructed from duplicate cores of tumor cells and surrounding stromal tissue from each resected specimen. Immunohistochemistry was used to evaluate the expression of each molecular marker. Results: In univariate analyses, high tumor cell expression of VEGF-A (P = .0005), VEGFR-1 (P = .013), VEGFR-2 (P = .006) and VEGFR-3 (P = .0003), were negative prognostic indicators for disease-specific survival (DSS). The most significant correlations between angiogenic marker expression and DSS were observed in patients with T2 stage and/or with sqamous cell carcinomas. In tumor stroma, however, high expression of VEGF-A (P = .017), VEGF-C (P = .003), VEGF-D (P = .009), VEGFR-1 (P = .01) and VEGFR-2 (P = .019), correlated with good prognosis. In multivariate analyses, high expression in tumor cells of VEGFR-3 (P = .007) was an independent negative prognostic factor for DSS, whereas high VEGF-C (P = .004) expression in stromal cells had an independent positive impact on survival. Conclusions: While high tumor cell expression of VEGFR-3 is an independent predictor of reduced survival in primary NSCLC, high VEGF-C expression in stromal cells is, in contrast, a favorable independent prognostic indicator. No significant financial relationships to disclose.