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High-grade serous ovarian cancer (HGSC) is the most prevalent and lethal ovarian cancer type. While HGSC usually responds well to primary treatment, most cases eventually relapse. Functional precision medicine - tailoring individualized treatments based on functional in vitro assays on patient-derived cells - has been recently employed in cancer clinical trials. Cancer organoids - three-dimensional, self-organizing, self-renewing cell cultures that recapitulate original tissue structure and function - have been applied as cellular models in these trials. However, in case of HGSC, organoid derivation has proven time consuming and inefficient, hindering their application in functional precision medicine due to a short time window, in which therapy for each patient needs to be selected. To address this problem, we aimed to establish whether drug vulnerabilities at HGSC relapse could be predicted using organoids derived from the primary disease cells. We derived sequential organoid models from material sampled during primary treatment and at relapse. Then, for organoid pairs (primary-relapse), we performed large-scale drug response profiling of a library of 370 compounds (approved drugs or drugs in clinical development), in 384-well microplate format, alone or in combination with a standard HGSC chemotherapeutic agent carboplatin. First, we found that HGSC organoid responses to standard chemotherapeutics retrospectively correlated to observed clinical treatment outcomes. But further, for each patient we identified compounds with pronounced cytotoxicity both in the primary and in the relapsed model, amounting to 66% of all hits (7% were primary-specific and 27% relapse-specific). We then focused on identifying patient-specific hits rather than compounds displaying general toxicity in all patient models. Based on a potential clinical applicability, for three patients we selected compounds for validation in organoid outgrowth assay, with prolonged (>1 month) drug-free period post-treatment. In two patients, AZD4573, a selective CDK9 inhibitor in clinical development for hematological malignancies, at nanomolar concentrations caused eradication of organoids when combined with carboplatin. Organoids from the third patient were vulnerable to nitazoxanide, an approved anti-helminthic agent and an inhibitor of mitochondrial oxidative phosphorylation. Importantly, the selected final hits were identified solely based on screening in organoid models from primary disease. In summary, we here demonstrate that HGSC organoids derived from primary disease material predict a majority of patient-specific drug vulnerabilities of organoids derived from the relapsed HGSC lesions. This indicates that patient stratification in functional precision medicine for treatment of HGSC relapse could be prospectively performed at the primary disease stage. Citation Format: Wojciech Senkowski, Laura Gall-Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Yan-Jun Chen, Erdogan P. Erkan, Mia K. Høg, Ida M. Larsen, Tarja Lamminen, Katja Kaipio, Jutta Huvila, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. A platform utilizing high-grade serous ovarian cancer organoids for prospective patient stratification in functional precision medicine. [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 5779.

Venetoclax has changed the clinical outlook for elderly and unfit patients with acute myeloid leukemia, but development of resistance is a challenge. In this issue of Cancer Cell, Bhatt et al. provide a general mechanism for how resistance emerges but also indications for how venetoclax-resistant cases may be treated.

Extensive utilization of organoids from high-grade serous ovarian carcinoma (HGSOC), the most common and lethal ovarian cancer, has been hampered by low success rates of long-term culture and scarcity of fresh tumor material. Here we present the development of a novel method for efficient generation, expansion and use of HGSOC organoids from cryopreserved tumor material. First, we assessed commonly used organoid media components and found that supplements such as FGF-2, R-Spondin1, Wnt or Noggin had negative impact on the HGSOC organoid derivation. But further, we found that supplementation with FGF-4, which has not been used in cancer organoid culture before, is beneficial for HGSOC organoid growth. Through extensive testing of various supplements and their combinations, we designed two novel HGSOC organoid media formulations - Medium 1 (M1) and Medium 2 (M2). Using M1 and M2 enabled generation and long-term expansion of living HGSOC organoid biobank with markedly improved success rate than in previous reports (55% vs. 23-38%). The organoids were established from cryopreserved tumor material, demonstrating the feasibility of using frozen tissue biobanks for HGSOC organoid derivation. Overall, we generated a collection of 18 expandable HGSOC organoid lines from 11 patients, encompassing samples from different tissue sites and disease progression stages. We validated the organoids using whole-genome sequencing, immunohistochemistry and single-cell RNA sequencing and demonstrated that they are genetically and phenotypically representative of original patient samples over long-term culture. Based on available patient consents, we deposited 3 organoid lines in a publicly accessible biobank. Finally, we investigated whether organoid drug responses correlate to those observed earlier in the clinic in the corresponding patients. Organoid-based drug-response profiling of clinically used HGSOC drug collection was performed in 384-well microplate format. To explore whether growth conditions impact correlation between organoid drug responses and clinical response, we compared the organoid drug responses in the nutrient-rich M1/M2 growth media to the ones observed in human plasma-like medium (HPLM), supplemented with relevant niche factors from M1/M2. Organoid drug responses correlated with clinical treatment outcomes, but only for organoids maintained in HPLM (Spearman r = 0.987, p=0.007 in HPLM vs 0.607, p=0.167 in growth medium, n=7), highlighting the importance of culture conditions in organoid-based functional assays. Taken together, we introduce a resource for efficient development and use of HGSOC organoids from cryopreserved material in ovarian cancer research. Citation Format: Wojciech Senkowski, Laura Gall Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Erdogan P. Erkan, Terese K. Høj, Mia K. Høg, Tarja Lamminen, Katja Kaipio, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni-Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. Efficient establishment and utilization of a high-grade serous ovarian cancer organoid biobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3069.

C4.4A is a glycosylphosphatidylinositol-anchored membrane protein comprised of two LU domains (Ly6/uPAR-like domains) and an extensively O-glycosylated C-terminal Ser/Thr/Pro-rich region. C4.4A is a novel biomarker for squamous epithelial differentiation. Its expression is dysregulated under various pathological conditions and it is a robust biomarker for poor prognosis in various malignant conditions such as pulmonary adenocarcinoma. To facilitate crystallization, the two LU domains were excised from intact C4.4A by limited proteolysis, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to 2.7 Å resolution and belonged to space groupC2221, with unit-cell parametersa= 55.49,b= 119.63,c= 168.54 Å. The statistics indicated good quality of the data, which form a solid basis for the determination of the C4.4A structure.

Ly6/uPAR/α-neurotoxin domain (LU-domain) is characterized by the presence of 4-5 disulfide bonds and three flexible loops that extend from a core stacked by several conversed disulfide bonds (thus also named three-fingered protein domain). This highly structurally stable protein domain is typically a protein-binder at extracellular space. Most LU proteins contain only single LU-domain as represented by Ly6 proteins in immunology and α-neurotoxins in snake venom. For Ly6 proteins, many are expressed in specific cell lineages and in differentiation stages, and are used as markers. In this study, we report the crystal structures of the two LU-domains of human C4.4A alone and its complex with a Fab fragment of a monoclonal anti-C4.4A antibody. Interestingly, both structures showed that C4.4A forms a very compact globule with two LU-domain packed face to face. This is in contrast to the flexible nature of most LU-domain-containing proteins in mammals. The Fab combining site of C4.4A involves both LU-domains, and appears to be the binding site for AGR2, a reported ligand of C4.4A. This work reports the first structure that contain two LU-domains and provides insights on how LU-domains fold into a compact protein and interacts with ligands.

SUMMARY Itch is an unpleasant skin sensation that can be triggered by exposure to many chemicals, including those released by mast cells. The natriuretic polypep-tide b (Nppb)-expressing class of sensory neurons, when activated, elicits scratching responses in mice, but it is unclear which itch-inducing agents stimulate these cells and the receptors involved. Here, we identify receptors expressed by Nppb neurons and demonstrate the functional importance of these receptors as sensors of endogenous pruritogens released by mast cells. Our search for receptors in Nppb neurons reveals that they express leukotriene, serotonin, and sphingosine-1-phosphate receptors. Targeted cell ablation, calcium imaging of primary sensory neurons, and conditional receptor knockout studies demonstrate that these receptors induce itch by the direct stimulation of Nppb neurons and neurotransmission through the canonical gastrin-releasing peptide (GRP)-dependent spinal cord itch pathway. Together, our results define a molecular and cellular pathway for mast cell-induced itch., Graphical Abstract, In Brief Solinski et al. describe a cellular and molecular pathway that couples mast cell activation to itch behavior. They show that mast cell mediators, serotonin, leukotriene-C4, and sphingosine-1 phosphate directly stimulate Nppb sensory neurons via Htr1f, Cysltr2, and S1pr1 receptors, and this results in itch responses that are elicited through a canonical spinal cord-GRP pathway.

Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide.

C4.4A and Haldisin belong to the Ly6/uPAR/α-neurotoxin protein domain family. They exhibit highly regulated expression profiles in normal epidermis, where they are confined to early (C4.4A) and late (Haldisin) squamous differentiation. We have now explored if dysregulated expressions occur in non-invasive and invasive skin lesions. In non-invasive lesions, their expression signatures were largely maintained as defined by that of normal epidermis. The scenario was, however, markedly different in the progression towards invasive squamous cell carcinomas. In its non-invasive stage (carcinoma in situ), a pronounced attenuation of C4.4A expression was observed, but upon transition to malignant invasive squamous cell carcinomas, the invasive fronts regained high expression of C4.4A. A similar progression was observed for the early stages of benign infiltrating keratoacanthomas. Interestingly, this transition was accompanied by a shift in the predominant association of C4.4A expression with CK1/10 in the normal epidermis to CK5/14 in the invasive lesions. In contrast, Haldisin expression maintained its confinement to the most-differentiated cells and was hardly expressed in the invasive lesions. Because this altered expression of C4.4A was seen in the invasive front of benign (keratoacanthomas) and malignant (squamous cell carcinomas) neoplasms, we propose that this transition of expression is primarily related to the invasive process.

C4.4A is a modular glycolipid-anchored Ly6/uPAR/α-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein with an established role in focalizing uPA-mediated plasminogen activation on cell surfaces. Distinct from this function, uPAR also modulates cell adhesion and migration on vitronectin-rich matrices. Although uPA and vitronectin engage structurally distinct binding sites on uPAR, they nonetheless cooperate functionally, as uPA binding potentiates uPAR-dependent induction of lamellipodia on vitronectin matrices. We now present data advancing the possibility that it is the burial of the β-hairpin in uPA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized by engagement of the β-hairpin in uPA, favors the proper assembly of an active, compact receptor structure that stimulates lamellipodia induction on vitronectin. This molecular model has wide implications for drug development targeting uPAR function.

In the version of this article initially published online, the labels were switched for the right-hand pair of bars in Fig. 4e. The left one of the two should be Chloroquine + veh, the right one Chloroquine + CNO. The error has been corrected in the print, HTML and PDF versions of the article.

Several members of the Ly-6/uPAR (LU)-protein domain family are differentially expressed in human squamous epithelia. In some cases, they even play important roles in maintaining skin homeostasis, as exemplified by the secreted single domain member, SLURP-1, the deficiency of which is associated with the development of palmoplantar hyperkeratosis in the congenital skin disorder Mal de Meleda. In the present study, we have characterized a new member of the LU-protein domain family, which we find to be predominantly expressed in the stratum granulosum of human skin, thus resembling the expression of SLURP-1. In accordance with its expression pattern, we denote this protein product, which is encoded by the LYPD5 gene, as Haldisin (human antigen with LU-domains expressed in skin). Two of the five human glycolipid-anchored membrane proteins with multiple LU-domains characterized so far are predominantly confined to squamous epithelia (i.e., C4.4A), to stratum spinosum, and Haldisin to stratum granulosum under normal homeostatic conditions. Whether Haldisin is a prognostic biomarker for certain epithelial malignancies, like C4.4A and SLURP-1, remains to be explored.

The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited by uPA binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexibility by covalently tethering domains DI and DIII via a non-natural interdomain disulfide bond (uPAR(H47C-N259C)). The corresponding soluble receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered binding kinetics towards uPA. Most importantly, the purified uPAR(H47C-N259C) also displays a gain in affinity for the somatomedin B domain of vitronectin compared with uPAR(wt), thus recapitulating the improved affinity that accompanies uPA-uPAR(wt) complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus bypasses the regulatory role of uPA binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.

The protein C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor, is a potential new biomarker in non-small cell lung cancer, with high levels of expression recently shown to correlate to poor survival of adenocarcinoma patients. In this study, C4.4A immunoreactivity in precursor lesions of lung squamous cell carcinoma and adenocarcinoma was investigated by stainings with a specific anti-C4.4A antibody. In the transformation from normal bronchial epithelium to squamous cell carcinoma, C4.4A was weakly expressed in basal cell hyperplasia but dramatically increased in squamous metaplasia. This was confined to the cell membrane and sustained in dysplasia, carcinoma in situ, and the invasive carcinoma. The induction of C4.4A already at the stage of hyperplasia could indicate that it is a marker of very early squamous differentiation, which aligns well with our earlier finding that C4.4A expression levels do not provide prognostic information on the survival of squamous cell carcinoma patients. In the progression from normal alveolar epithelium to peripheral adenocarcinoma, we observed an unexpected, distinct cytoplasmic staining for C4.4A in a fraction of atypical adenomatous hyperplasias, while most bronchioloalveolar carcinomas were negative. Likewise, only a fraction of the invasive adenocarcinomas was positive for C4.4A. With a view to the prognostic impact of C4.4A in adenocarcinoma patients, this finding might suggest that C4.4A could be an early biomarker for a possibly more malignant subtype of this disease.

The glycosylphosphatidylinositol (GPI)–anchored C4.4A was originally identified as a metastasis-associated protein by differential screening of rat pancreatic carcinoma cell lines. C4.4A is accordingly expressed in various human carcinoma lesions. Although C4.4A is a structural homolog of the urokinase receptor (uPAR), which is implicated in cancer invasion and metastasis, no function has so far been assigned to C4.4A. To assist future studies on its function in both physiological and pathophysiological conditions, the present study provide a global survey on C4.4A expression in the normal mouse by a comprehensive immunohistochemical mapping. This task was accomplished by staining paraffin-embedded tissues with a specific rabbit polyclonal anti-C4.4A antibody. In the adult mouse, C4.4A was predominantly expressed in the suprabasal layers of the squamous epithelia of the oral cavity, esophagus, non-glandular portion of the rodent stomach, anus, vagina, cornea, and skin. This epithelial confinement was particularly evident from the abrupt termination of C4.4A expression at the squamo-columnar transition zones found at the ano-rectal and utero-vaginal junctions, for example. During mouse embryogenesis, C4.4A expression first appears in the developing squamous epithelium at embryonic day 13.5. This anatomical location of C4.4A is thus concordant with a possible functional role in early differentiation of stratified squamous epithelia.

In the last two decades, the urokinase-type plasminogen activator receptor (uPAR) has been implicated in a number of human pathologies such as cancer, bacterial infections, and paroxysmal nocturnal hemoglobinuria. The primary function of this glycolipid-anchored receptor is to focalize uPA-mediated plasminogen activation at the cell surface, which is accomplished by its high-affinity interaction with the growth factor-like domain of uPA. Detailed insights into the molecular basis underlying the interactions between uPAR and its two bona fide ligands, uPA and vitronectin, have been obtained recently by X-ray crystallography and surface plasmon resonance studies. Importantly, these structural studies also define possible druggable target sites in uPAR for small molecules and provide guidelines for the development of reporter groups applicable for non-invasive molecular imaging of uPAR expression in vivo by positron emission tomography. In this review, we will discuss recent advances in our perception of the structure-function relationships of uPAR ligation and how these may assist translational research in preclinical intervention studies of uPAR function.

The high prevalence and mortality of lung cancer, together with a poor 5-year survival of only approximately 15%, emphasize the need for prognostic and predictive factors to improve patient treatment. C4.4A, a member of the Ly6/uPAR family of membrane proteins, qualifies as such a potential informative biomarker in non-small cell lung cancer. Under normal physiological conditions, it is primarily expressed in suprabasal layers of stratified squamous epithelia. Consequently, it is absent from healthy bronchial and alveolar tissue, but nevertheless appears at early stages in the progression to invasive carcinomas of the lung, i.e., in bronchial hyperplasia/metaplasia and atypical adenomatous hyperplasia. In the stages leading to pulmonary squamous cell carcinoma, expression is sustained in dysplasia, carcinoma in situ and invasive carcinomas, and this pertains to the normal presence of C4.4A in squamous epithelium. In pulmonary adenocarcinomas, a fraction of cases is positive for C4.4A, which is surprising, given the origin of these carcinomas from mucin-producing and not squamous epithelium. Interestingly, this correlates with a highly compromised patient survival and a predominant solid tumor growth pattern. Circumstantial evidence suggests an inverse relationship between C4.4A and the tumor suppressor LKB1. This might provide a link to the prognostic impact of C4.4A in patients with adenocarcinomas of the lung and could potentially be exploited for predicting the efficacy of treatment targeting components of the LKB1 pathway.

The broad research use of organoids from high-grade serous ovarian carcinoma (HGSC) has been hampered by low culture success rates and limited availability of fresh tumor material. Here we describe a method for generation and long-term expansion of HGSC organoids with efficacy markedly improved over previous reports (55% vs. 23-38%). We established organoids from cryopreserved material, demonstrating the feasibility of using viably biobanked tissue for HGSC organoid derivation. Genomic, histologic and single-cell transcriptomic analyses revealed that organoids recapitulated genetic and phenotypic features of original tumors. Organoid drug responses correlated with clinical treatment outcomes, although in culture conditions-dependent manner and only in organoids maintained in human plasma-like medium (HPLM). Organoids from consenting patients are available to the research community through a public biobank and organoid genomic data explorable through an interactive online tool. Taken together, this resource facilitates the application of HGSC organoids in basic and translational ovarian cancer research.