Hadeesha Piyadasa

All Publications

• Temporal and spatial composition of the tumor microenvironment predicts response to immune checkpoint inhibition. bioRxiv : the preprint server for biology Greenwald, N. F., Nederlof, I., Sowers, C., Ding, D. Y., Park, S., Kong, A., Houlahan, K. E., Varra, S. R., de Graaf, M., Geurts, V., Liu, C. C., Ranek, J. S., Voorwerk, L., de Maaker, M., Kagel, A., McCaffrey, E., Khan, A., Yeh, C. Y., Fullaway, C. C., Khair, Z., Bai, Y., Piyadasa, H., Risom, T., Delmastro, A., Hartmann, F. J., Mangiante, L., Sotomayor-Vivas, C., Schumacher, T. N., Ma, Z., Bosse, M., van de Vijver, M. J., Tibshirani, R., Horlings, H. M., Curtis, C., Kok, M., Angelo, M. 2025

  Abstract

  Immune checkpoint inhibition (ICI) has fundamentally changed cancer treatment. However, only a minority of patients with metastatic triple negative breast cancer (TNBC) benefit from ICI, and the determinants of response remain largely unknown. To better understand the factors influencing patient outcome, we assembled a longitudinal cohort with tissue from multiple timepoints, including primary tumor, pre-treatment metastatic tumor, and on-treatment metastatic tumor from 117 patients treated with ICI (nivolumab) in the phase II TONIC trial. We used highly multiplexed imaging to quantify the subcellular localization of 37 proteins in each tumor. To extract meaningful information from the imaging data, we developed SpaceCat, a computational pipeline that quantifies features from imaging data such as cell density, cell diversity, spatial structure, and functional marker expression. We applied SpaceCat to 678 images from 294 tumors, generating more than 800 distinct features per tumor. Spatial features were more predictive of patient outcome, including features like the degree of mixing between cancer and immune cells, the diversity of the neighboring immune cells surrounding cancer cells, and the degree of T cell infiltration at the tumor border. Non-spatial features, including the ratio between T cell subsets and cancer cells and PD-L1 levels on myeloid cells, were also associated with patient outcome. Surprisingly, we did not identify robust predictors of response in the primary tumors. In contrast, the metastatic tumors had numerous features which predicted response. Some of these features, such as the cellular diversity at the tumor border, were shared across timepoints, but many of the features, such as T cell infiltration at the tumor border, were predictive of response at only a single timepoint. We trained multivariate models on all of the features in the dataset, finding that we could accurately predict patient outcome from the pre-treatment metastatic tumors, with improved performance using the on-treatment tumors. We validated our findings in matched bulk RNA-seq data, finding the most informative features from the on-treatment samples. Our study highlights the importance of profiling sequential tumor biopsies to understand the evolution of the tumor microenvironment, elucidating the temporal and spatial dynamics underlying patient responses and underscoring the need for further research on the prognostic role of metastatic tissue and its utility in stratifying patients for ICI.

  View details for DOI 10.1101/2025.01.26.634557

  View details for PubMedID 39975273

  View details for PubMedCentralID PMC11838242

• Automated classification of cellular expression in multiplexed imaging data with Nimbus. bioRxiv : the preprint server for biology Rumberger, J. L., Greenwald, N. F., Ranek, J. S., Boonrat, P., Walker, C., Franzen, J., Varra, S. R., Kong, A., Sowers, C., Liu, C. C., Averbukh, I., Piyadasa, H., Vanguri, R., Nederlof, I., Wang, X. J., Van Valen, D., Kok, M., Hollmann, T. J., Kainmueller, D., Angelo, M. 2024

  Abstract

  Multiplexed imaging offers a powerful approach to characterize the spatial topography of tissues in both health and disease. To analyze such data, the specific combination of markers that are present in each cell must be enumerated to enable accurate phenotyping, a process that often relies on unsupervised clustering. We constructed the Pan-Multiplex (Pan-M) dataset containing 197 million distinct annotations of marker expression across 15 different cell types. We used Pan-M to create Nimbus, a deep learning model to predict marker positivity from multiplexed image data. Nimbus is a pre-trained model that uses the underlying images to classify marker expression across distinct cell types, from different tissues, acquired using different microscope platforms, without requiring any retraining. We demonstrate that Nimbus predictions capture the underlying staining patterns of the full diversity of markers present in Pan-M. We then show how Nimbus predictions can be integrated with downstream clustering algorithms to robustly identify cell subtypes in image data. We have open-sourced Nimbus and Pan-M to enable community use at https://github.com/angelolab/Nimbus-Inference.

  View details for DOI 10.1101/2024.06.02.597062

  View details for PubMedID 38895405

  View details for PubMedCentralID PMC11185540

• Rapid Setup of Tissue Microarray and Tiled Area Imaging on the Multiplexed Ion Beam Imaging Microscope using the Tile/SED/Array Interface. Journal of visualized experiments : JoVE Piyadasa, H., Oberlton, B., Kong, A., Camacho Fullaway, C., Reddy Varra, S., Sowers, C., Tsai, A. G. 2023

  Abstract

  Multiplexed ion beam imaging (MIBI) is a next-generation mass spectrometry-based microscopy technique that generates 40+ plex images of protein expression in histologic tissues, enabling detailed dissection of cellular phenotypes and histoarchitectural organization. A key bottleneck in operation occurs when users select the physical locations on the tissue for imaging. As the scale and complexity of MIBI experiments have increased, the manufacturer-provided interface and third-party tools have become increasingly unwieldy for imaging large tissue microarrays and tiled tissue areas. Thus, a web-based, interactive, what-you-see-is-what-you-get (WYSIWYG) graphical interface layer - the tile/SED/array Interface (TSAI) - was developed for users to set imaging locations using familiar and intuitive mouse gestures such as drag-and-drop, click-and-drag, and polygon drawing. Written according to web standards already built into modern web browsers, it requires no installation of external programs, extensions, or compilers. Of interest to the hundreds of current MIBI users, this interface dramatically simplifies and accelerates the setup of large, complex MIBI runs.

  View details for DOI 10.3791/65615

  View details for PubMedID 37782085

• A spatially resolved timeline of the human maternal-fetal interface. Nature Greenbaum, S., Averbukh, I., Soon, E., Rizzuto, G., Baranski, A., Greenwald, N. F., Kagel, A., Bosse, M., Jaswa, E. G., Khair, Z., Kwok, S., Warshawsky, S., Piyadasa, H., Goldston, M., Spence, A., Miller, G., Schwartz, M., Graf, W., Van Valen, D., Winn, V. D., Hollmann, T., Keren, L., van de Rijn, M., Angelo, M. 2023; 619 (7970): 595-605

  Abstract

  Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR)1-3. However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells.

  View details for DOI 10.1038/s41586-023-06298-9

  View details for PubMedID 37468587

  View details for PubMedCentralID PMC10356615

• Spatial proteomics of tumor microenvironments reveal why location matters. Nature immunology Piyadasa, H., Angelo, M., Bendall, S. C. 2023

  View details for DOI 10.1038/s41590-023-01471-8

  View details for PubMedID 36959293

  View details for PubMedCentralID 5998822

• Combination of IL-17A/F and TNF-alpha uniquely alters the bronchial epithelial cell proteome to enhance proteins that augment neutrophil migration. Journal of inflammation (London, England) Altieri, A., Piyadasa, H., Hemshekhar, M., Osawa, N., Recksiedler, B., Spicer, V., Hiemstra, P. S., Halayko, A. J., Mookherjee, N. 2022; 19 (1): 26

  Abstract

  BACKGROUND: The heterodimer interleukin (IL)-17A/F is elevated in the lungs in chronic respiratory disease such as severe asthma, along with the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Although IL-17A/F and TNF-alpha are known to functionally cooperate to exacerbate airway inflammation, proteins altered by their interaction in the lungs are not fully elucidated.RESULTS: We used Slow Off-rate Modified Aptamer-based proteomic array to identify proteins that are uniquely and/or synergistically enhanced by concurrent stimulation with IL-17A/F and TNF-alpha in human bronchial epithelial cells (HBEC). The abundance of 38 proteins was significantly enhanced by the combination of IL-17A/F and TNF-alpha, compared to either cytokine alone. Four out of seven proteins that were increased>2-fold were those that promote neutrophil migration; host defence peptides (HDP; Lipocalin-2 (LCN-2) and Elafin) and chemokines (IL-8, GROalpha). We independently confirmed the synergistic increase of these four proteins by western blots and ELISA. We also functionally confirmed that factors secreted by HBEC stimulated with the combination of IL-17A/F and TNF-alpha uniquely enhances neutrophil migration. We further showed that PI3K and PKC pathways selectively control IL-17A/F+TNF-alpha-mediated synergistic production of HDPs LCN-2 and Elafin, but not chemokines IL-8 and GROalpha. Using a murine model of airway inflammation, we demonstrated enhancement of IL-17A/F, TNF-alpha, LCN-2 and neutrophil chemokine KC in the lungs, thus corroborating our findings in-vivo.CONCLUSION: This study identifies proteins and signaling mediated by concurrent IL-17A/F and TNF-alpha exposure in the lungs, relevant to respiratory diseases characterized by chronic inflammation, especially neutrophilic airway inflammation such as severe asthma.

  View details for DOI 10.1186/s12950-022-00323-w

  View details for PubMedID 36517803

• Characterization of sex-related differences in allergen house dust mite-challenged airway inflammation, in two different strains of mice. Scientific reports Mostafa, D. H., Hemshekhar, M., Piyadasa, H., Altieri, A., Halayko, A. J., Pascoe, C. D., Mookherjee, N. 2022; 12 (1): 20837

  Abstract

  Biological sex impacts disease prevalence, severity and response to therapy in asthma, however preclinical studies often use only one sex in murine models. Here, we detail sex-related differences in immune responses using a house dust mite (HDM)-challenge model of acute airway inflammation, in adult mice of two different strains (BALB/c and C57BL/6NJ). Female and male mice were challenged (intranasally) with HDM extract (~25mug) for 2weeks (N=10 per group). Increase in serum HDM-specific IgE showed a female bias, which was statistically significant in BALB/c mice. We compared naive and HDM-challenged mice to define immune responses in the lungs by assessing leukocyte accumulation in the bronchoalveolar lavage fluid (BALF), and profiling the abundance of 29 different cytokines in BALF and lung tissue lysates. Our results demonstrate specific sex-related and strain-dependent differences in airway inflammation. For example, HDM-driven accumulation of neutrophils, eosinophils and macrophages were significantly higher in females compared to males, in BALB/c mice. In contrast, HDM-mediated eosinophil accumulation was higher in males compared to females, in C57BL/6NJ mice. Differences in lung cytokine profiles indicated that HDM drives a T-helper (Th)17-biased response with higher IL-17 levels in female BALB/c mice compared to males, whereas female C57BL/6NJ mice elicit a mixed Th1/Th2-skewed response. Male mice of both strains showed higher levels of specific Th2-skewed cytokines, such as IL-21, IL-25 and IL-9, in response to HDM. Overall, this study details sex dimorphism in HDM-mediated airway inflammation in mice, which will be a valuable resource for preclinical studies in allergic airway inflammation and asthma.

  View details for DOI 10.1038/s41598-022-25327-7

  View details for PubMedID 36460835

• Rodent Lung Tissue Sample Preparation and Processing for Shotgun Proteomics. Methods in molecular biology (Clifton, N.J.) Piyadasa, H., Lao, Y., Krokhin, O., Mookherjee, N. 2022; 2456: 53-62

  Abstract

  Mass spectrometry (MS) is a routinely used approach to characterize global protein profile in various biological samples. Here we describe rodent lung tissue homogenization, sample preparation, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for shotgun proteomics.

  View details for DOI 10.1007/978-1-0716-2124-0_4

  View details for PubMedID 35612734

• Sex Dimorphism of Allergen-Induced Secreted Proteins in Murine and Human Lungs. Frontiers in immunology Hemshekhar, M., Mostafa, D. H., Spicer, V., Piyadasa, H., Maestre-Batlle, D., Bolling, A. K., Halayko, A. J., Carlsten, C., Mookherjee, N. 2022; 13: 923986

  Abstract

  Biological sex influences disease severity, prevalence and response to therapy in allergic asthma. However, allergen-mediated sex-specific changes in lung protein biomarkers remain undefined. Here, we report sex-related differences in specific proteins secreted in the lungs of both mice and humans, in response to inhaled allergens. Female and male BALB/c mice (7-8 weeks) were intranasally challenged with the allergen house dust mite (HDM) for 2 weeks. Bronchoalveolar lavage fluid (BALF) was collected 24 hour after the last HDM challenge from allergen-naive and HDM-challenged mice (N=10 per group, each sex). In a human study, adult participants were exposed to nebulized (2min) allergens (based on individual sensitivity), BALF was obtained after 24 hour (N=5 each female and male). The BALF samples were examined in immunoblots for the abundance of 10 proteins shown to increase in response to allergen in both murine and human BALF, selected from proteomics studies. We showed significant sex-bias in allergen-driven increase in five out of the 10 selected proteins. Of these, increase in eosinophil peroxidase (EPX) was significantly higher in females compared to males, in both mice and human BALF. We also showed specific sex-related differences between murine and human samples. For example, allergen-driven increase in S100A8 and S100A9 was significantly higher in BALF of females compared to males in mice, but significantly higher in males compared to females in humans. Overall, this study provides sex-specific protein biomarkers that are enhanced in response to allergen in murine and human lungs, informing and motivating translational research in allergic asthma.

  View details for DOI 10.3389/fimmu.2022.923986

  View details for PubMedID 35837410

• Disrupting Tryptophan in the Central Hydrophobic Region Selectively Mitigates Immunomodulatory Activities of the Innate Defence Regulator Peptide IDR-1002. Journal of medicinal chemistry Piyadasa, H., Hemshekhar, M., Osawa, N., Lloyd, D., Altieri, A., Basu, S., Krokhin, O. V., Halayko, A. J., Mookherjee, N. 2021

  Abstract

  Innate defense regulator (IDR) peptides show promise as immunomodulatory therapeutics. However, there is limited understanding of the relationship of IDR peptide sequence and/or structure with its immunomodulatory activity. We previously reported that an IDR peptide, IDR-1002, reduces airway hyperresponsiveness (AHR) and inflammation in a house dust mite (HDM)-challenged murine model of airway inflammation. Here, we examined the sequence-to-function relationship of IDR-1002 in HDM-challenged mice and human bronchial epithelial cells (HBEC). We demonstrated that the tryptophan (W8) in the central hydrophobic region of IDR-1002 is required for the peptide to (i) suppress the pro-inflammatory cytokine IL-33, and induce anti-inflammatory mediators IL-1RA and stanniocalcin-1 in HBEC, and (ii) reduce IL-33 abundance, and eosinophil and neutrophil infiltration, in the lungs of HDM-challenged mice, without affecting the capacity to improve AHR, suggesting multimodal activity in vivo. Findings from this study can be used to design IDR peptides with targeted impact on immunomodulation and pathophysiology in respiratory diseases.

  View details for DOI 10.1021/acs.jmedchem.0c02065

  View details for PubMedID 33974425