Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays

Finetti, Francesca; Capitani, Nagaja; Manganaro, Noemi; Tatangelo, Vanessa; Libonati, Francesca; Panattoni, Giulia; Calaresu, Ivo; Ballerini, Laura; Baldari, Cosima T.; Patrussi, Laura

doi:10.3389/fimmu.2020.00471

Cited by 9 publications

(8 citation statements)

References 26 publications

(30 reference statements)

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“…3F), which develop an aggressive disease [25]. Similar results were obtained using organotypic cultures from spleens of wild-type mice, a system that recapitulates the key physiological features of lymphoid organs [26], cultured in media conditioned by B cells from wild-type, E-TCL1 or E-TCL1/p66Shc -/mice (Fig. 3G).…”

Section: P66shc Deficiency In Leukemic Cells From Eμ-tcl1 Mice Enhanc...supporting

confidence: 72%

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia

Boncompagni,

Tatangelo,

Lopresti

et al. 2023

Preprint

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The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eu-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adaptor whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eu-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eu-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

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Section: P66shc Deficiency In Leukemic Cells From Eμ-tcl1 Mice Enhanc...supporting

confidence: 72%

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia

Boncompagni,

Tatangelo,

Lopresti

et al. 2023

Preprint

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“…The results were translated to organotypic cultures of vibratome-generated 230-m spleen slices, where stromal cells remain in their 3D context and which maintain their architecture for over 48 h without significant viability loss ( Fig.S5A). 22 Homing chemokine mRNAs were quantified in slices cultured for 48 h with conditioned media from wild-type B cells or leukemic cells from E-TCL1 or E-TCL1/p66Shc -/mice. Consistent with the results obtained using OP9 cells, conditioned media from wild-type B cells affected to a minor extent chemokine expression in organotypic cultures ( Fig.2A), with an upregulation by conditioned media from leukemic E-TCL1 cells ( Fig.2A,S3C) that was further enhanced by leukemic E-TCL1/p66Shc -/cell-derived supernatants ( Fig.2A) (but for CXCL12, Fig.S3C).…”

Section: Results P66shc Deficiency Enhances the Ability Of Leukemic Ementioning

confidence: 99%

“…Vibratome-generated 230-m spleen slices, obtained as described, 22 were cultured at 37°C for 48 h either in high-glucose DMEM 7.5% BCS at 37°C, or in 100 l leukemic cell supernatant. For immunofluorescence microscopy spleen slices were transferred to 10well diagnostic microscope slides, fixed and permeabilized, washed and stained with anti-CCL2 in combination with anti-ER-TR7, anti-podoplanin, anti-CD3 or anti-CD19 Abs and fluorochrome-conjugated secondary antibodies (antibodies in Table S1).…”

Section: Organotypic Culture and Immunofluorescence Microscopy Of Splmentioning

confidence: 99%

Enhanced IL-9 secretion by p66Shc-deficient CLL cells modulates the chemokine landscape of the stromal microenvironment

Patrussi

Manganaro

Capitani

et al. 2021

Blood

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The stromal microenvironment is central to chronic lymphocytic leukemia (CLL) pathogenesis. How leukemic cells condition the stroma to enhance its chemoattractant properties remains elusive. Here we show that mouse and human CLL cells promote the contact-independent stromal expression of homing chemokines. This function was strongly enhanced in leukemic cells from Em-TCL1 mice lacking the pro-oxidant p66Shc adaptor, which develop an aggressive disease with organ infiltration. We identified Interleukin (IL) -9 as the soluble factor, negatively modulated by p66Shc, responsible for the chemokine-elevating activity of leukemic cells on stromal cells. IL-9 blockade in Em-TCL1/p66Shc-/- mice resulted in a decrease in the nodal expression of homing chemokines, which correlated with decreased leukemic cell invasiveness. IL-9 levels were found to inversely correlate with residual p66Shc in the p66Shc-deficient human CLL cells (n=52 patients). p66Shc reconstitution in CLL cells normalized IL-9 expression and neutralized their chemokine-elevating activity. Notably, high IL-9 expression in CLL cells directly correlates with lymphadenopathy, liver infiltration, disease severity and overall survival, emerging as an independent predictor of disease outcome. Our results demonstrate that IL-9 modulates the chemokine landscape in the stroma, and that p66Shc, by regulating IL-9 expression, tunes the ability of leukemic cells to shape the microenvironment, thereby contributing to CLL pathogenesis.

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“…Organotypic culture has previously been used for in vitro investigation of a range of tissues/cancers, including osteosarcoma [ 27 , 38 , 39 ], demonstrating its ability to preserve patients’ tumour/stromal cells, cytoarchitecture and both physiological and pathological settings. This organotypic culture method has also been used for screening cancer therapeutic agents [ 25 , 26 , 27 ], establishing its utility as a highly complex, reliable, cost-effective, and easy-to-handle method to study drug responses in vitro.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of the Chemotherapy Drug Response Using Organotypic Cultures of Osteosarcoma Tumours from Mice Models and Canine Patients

Brulin

Nolan

Marangon

et al. 2021

Cancers

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Improvements in the clinical outcome of osteosarcoma have plateaued in recent decades with poor translation between preclinical testing and clinical efficacy. Organotypic cultures retain key features of patient tumours, such as a myriad of cell types organized within an extracellular matrix, thereby presenting a more realistic and personalised screening of chemotherapeutic agents ex vivo. To test this concept for the first time in osteosarcoma, murine and canine osteosarcoma organotypic models were maintained for up to 21 days and in-depth analysis identified proportions of immune and stromal cells present at levels comparable to that reported in vivo in the literature. Cytotoxicity testing of a range of chemotherapeutic drugs (mafosfamide, cisplatin, methotrexate, etoposide, and doxorubicin) on murine organotypic culture ex vivo found limited response to treatment, with immune and stromal cells demonstrating enhanced survival over the global tumour cell population. Furthermore, significantly decreased sensitivity to a range of chemotherapeutics in 3D organotypic culture relative to 2D monolayer was observed, with subsequent investigation confirming reduced sensitivity in 3D than in 2D, even at equivalent levels of drug uptake. Finally, as proof of concept for the application of this model to personalised drug screening, chemotherapy testing with doxorubicin was performed on biopsies obtained from canine osteosarcoma patients. Together, this study highlights the importance of recapitulating the 3D tumour multicellular microenvironment to better predict drug response and provides evidence for the utility and possibilities of organotypic culture for enhanced preclinical selection and evaluation of chemotherapeutics targeting osteosarcoma.

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Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays

Cited by 9 publications

References 26 publications

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia

Enhanced IL-9 secretion by p66Shc-deficient CLL cells modulates the chemokine landscape of the stromal microenvironment

Evaluation of the Chemotherapy Drug Response Using Organotypic Cultures of Osteosarcoma Tumours from Mice Models and Canine Patients

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