Live slow-frozen human tumor tissues viable for 2D, 3D, ex vivo cultures and single-cell RNAseq

Restivo, Gaetana; Tastanova, Aizhan; Balázs, Zsolt; Panebianco, Federica; Diepenbruck, Maren; Ercan, Caner; Preca, Bodgan-T.; Hafner, Jürg; Weber, Walter; Kurzeder, Christian; Vetter, Marcus; Soysal, Simone Münst; Beisel, Christian; Bentires‐Alj, Mohamed; Piscuoglio, Salvatore; Krauthammer, Michael; Levesque, Mitchell P.

doi:10.1038/s42003-022-04025-0

Cited by 5 publications

(5 citation statements)

References 33 publications

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“…However, the area where cryopreserved tissue excels is in downstream live tissue testing where viable cells are required [36]. Advances in precision medicine rely on patient-derived tissue to perform ex vivo drug testing, xenograft modeling, and 3D organoid modeling, and the most viable source of tissue is either fresh from the patient or through a slow freezing, cryopreservation method [37], as was used in this study.…”

Section: Discussionmentioning

confidence: 99%

DNA Quantity and Quality Comparisons between Cryopreserved and FFPE Tumors from Matched Pan-Cancer Samples

Okojie,

O’Neal,

Burr

et al. 2024

Current Oncology

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Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing in clinical cancer care comes almost universally from formalin-fixed, paraffin-embedded tissue (FFPE). As newer technologies emerge for DNA characterization that requires higher molecular weight DNA, it was necessary to compare the quality of DNA in terms of DNA length between FFPE and cryopreserved samples. We hypothesized that cryopreserved samples would yield higher quantity and superior quality DNA compared to FFPE samples. We analyzed DNA metrics by performing a head-to-head comparison between FFPE and cryopreserved samples from 38 human tumors representing various cancer types. DNA quantity and purity were measured by UV spectrophotometry, and DNA from cryopreserved tissue demonstrated a 4.2-fold increase in DNA yield per mg of tissue (p-value < 0.001). DNA quality was measured on a fragment microelectrophoresis analyzer, and again, DNA from cryopreserved tissue demonstrated a 223% increase in the DNA quality number and a 9-fold increase in DNA fragments > 40,000 bp (p-value < 0.0001). DNA from the cryopreserved tissues was superior to the DNA from FFPE samples in terms of DNA yield and quality.

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Section: Discussionmentioning

confidence: 99%

DNA Quantity and Quality Comparisons between Cryopreserved and FFPE Tumors from Matched Pan-Cancer Samples

Okojie,

O’Neal,

Burr

et al. 2024

Current Oncology

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“…Primary and metastatic melanoma flow-frozen human biopsies were obtained from the URPP biobank, Zurich. Collection, preparation, and freezing protocols of the fresh biopsies are outlined in ( 32 ). Informed consent had been obtained from all patients and all experiments were conducted according to the ethical rules of the Cantonal Ethic Committee of Zurich (Ethics form BASEC: 2014-0425).…”

Section: Methodsmentioning

confidence: 99%

Investigating the tumor-immune microenvironment through extracellular vesicles from frozen patient biopsies and 3D cultures

2023

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Melanomas are highly immunogenic tumors that have been shown to activate the immune response. Nonetheless, a significant portion of melanoma cases are either unresponsive to immunotherapy or relapsed due to acquired resistance. During melanomagenesis, melanoma and immune cells undergo immunomodulatory mechanisms that aid in immune resistance and evasion. The crosstalk within melanoma microenvironment is facilitated through the secretion of soluble factors, growth factors, cytokines, and chemokines. In addition, the release and uptake of secretory vesicles known as extracellular vesicles (EVs) play a key role in shaping the tumor microenvironment (TME). Melanoma-derived EVs have been implicated in immune suppression and escape, promoting tumor progression. In the context of cancer patients, EVs are usually isolated from biofluids such as serum, urine, and saliva. Nonetheless, this approach neglects the fact that biofluid-derived EVs reflect not only the tumor, but also include contributions from different organs and cell types. For that, isolating EVs from tissue samples allows for studying different cell populations resident at the tumor site, such as tumor-infiltrating lymphocytes and their secreted EVs, which play a central anti-tumor role. Herein, we outline the first instance of a method for EV isolation from frozen tissue samples at high purity and sensitivity that can be easily reproduced without the need for complicated isolation methods. Our method of processing the tissue not only circumvents the need for hard-to-acquire freshly isolated tissue samples, but also preserves EV surface proteins which allows for multiplex surface markers profiling. Tissue-derived EVs provide insight into the physiological role of EVs enrichment at tumor sites, which can be overlooked when studying circulating EVs coming from different sources. Tissue-derived EVs could be further characterized in terms of their genomics and proteomics to identify possible mechanisms for regulating the TME. Additionally, identified markers could be correlated to overall patient survival and disease progression for prognostic purposes.

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“…Both tumor samples and reference tissues were cut to a thickness of 3 µm and placed on the same slide before staining and acquisition. For CyTOF analysis, material remaining after pathological assessment were prepared as slow frozen material by the SKINTEGRITY.CH Biobank (Dermatology Department of the University Hospital Zürich) as recently described (37).…”

Section: Methodsmentioning

confidence: 99%

“…The authors declare no competing interests concerning the data presented in this manuscript. Hospital Zürich) as recently described (37).…”

Section: Declaration Of Interestsmentioning

confidence: 99%

Standardization of suspension and imaging mass cytometry readouts for clinical decision making

Casanova

Sivapatham

et al. 2023

Preprint

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Suspension and imaging mass cytometry are single-cell, proteomic-based methods used to characterize tissue composition and structure. Data assessing the consistency of these methods over an extended period of time are still sparse and are needed if mass cytometry-based methods are to be used clinically. Here, we present experimental and computational pipelines developed within the Tumor Profiler clinical study, an observational clinical trial assessing the relevance of cutting-edge technologies in guiding treatment decisions for advanced cancer patients. By using aliquots of frozen antibody panels, batch effects between independent experiments performed within a time frame of one year were minimized. The inclusion of well-characterized reference samples allowed us to assess and correct for batch effects. A systematic evaluation of a test tumor sample analyzed in each run showed that our batch correction approach consistently reduced signal variations. We provide an exemplary analysis of a representative patient sample including an overview of data provided to clinicians and potential treatment suggestions. This study demonstrates that standardized suspension and imaging mass cytometry measurements generate robust data that meet clinical requirements for reproducibility and provide oncologists with valuable insights on the biology of patient tumors.

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Live slow-frozen human tumor tissues viable for 2D, 3D, ex vivo cultures and single-cell RNAseq

Cited by 5 publications

References 33 publications

DNA Quantity and Quality Comparisons between Cryopreserved and FFPE Tumors from Matched Pan-Cancer Samples

DNA Quantity and Quality Comparisons between Cryopreserved and FFPE Tumors from Matched Pan-Cancer Samples

Investigating the tumor-immune microenvironment through extracellular vesicles from frozen patient biopsies and 3D cultures

Standardization of suspension and imaging mass cytometry readouts for clinical decision making

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